Analysis of Citrus Bioflavonoid Content and Dipeptidyl Peptidase-4 Inhibitory Potential of Commercially Available Supplements.

Citrus bioflavonoids are polyphenolic plant-derived pigments found in high levels in oranges, lemons, grapefruits and other citrus fruits. The three most abundant types of citrus bioflavonoids are hesperidin, naringenin and eriocitrin. Citrus bioflavonoids have long been known to possess powerful free radical-scavenging properties and cardioprotective effects. The study involved the analysis of 10 commercially available citrus bioflavonoid supplements from three different countries: Australia, the United States and Canada. The supplements were tested for their citrus bioflavonoid content which varied from 0.8 to 33.3% w/w. The daily bioflavonoid dose varied from 19 mg to 560 mg. Hesperidin was the major citrus bioflavonoid in nine out of ten supplements. One supplement was found to contain less than 10% of the quantity of rutin claimed to have been added. The DPP-4 inhibitory potential, compared through an estimation of rutin equivalence, ranged from 1.9 mg to 400 mg per day. This data highlights the variability between the supplements in their potential to inhibit DPP-4 for subsequent health benefits.

Chronic ß2 -adrenoceptor agonist treatment alters muscle proteome and functional adaptations induced by high intensity training in young men.

KEY POINTS: While several studies have investigated the effects of exercise training in human skeletal muscle and the chronic effect of ß2 -agonist treatment in rodent muscle, their effects on muscle proteome signature with related functional measures in humans are still incompletely understood. Herein we show that daily ß2 -agonist treatment attenuates training-induced enhancements in exercise performance and maximal oxygen consumption, and alters muscle proteome signature and phenotype in trained young men. Daily ß2 -agonist treatment abolished several of the training-induced enhancements in muscle oxidative capacity and caused a repression of muscle metabolic pathways; furthermore, ß2 -agonist treatment induced a slow-to-fast twitch muscle phenotype transition. The present study indicates that chronic ß2 -agonist treatment confounds the positive effect of high intensity training on exercise performance and oxidative capacity, which is of interest for the large proportion of persons using inhaled ß2 -agonists on a daily basis, including athletes. ABSTRACT: Although the effects of training have been studied for decades, data on muscle proteome signature remodelling induced by high intensity training in relation to functional changes in humans remains incomplete. Likewise, ß2 -agonists are frequently used to counteract exercise-induced bronchoconstriction, but the effects ß2 -agonist treatment on muscle remodelling and adaptations to training are unknown. In a placebo-controlled parallel study, we randomly assigned 21 trained men to 4 weeks of high intensity training with (HIT+ß2 A) or without (HIT) daily inhalation of ß2 -agonist (terbutaline, 4 mg dose-1 ). Of 486 proteins identified by mass-spectrometry proteomics of muscle biopsies sampled before and after the intervention, 32 and 85 were changing (false discovery rate (FDR) ≤5%) with the intervention in HIT and HIT+ß2 A, respectively. Proteome signature changes were different in HIT and HIT+ß2 A (P = 0.005), wherein ß2 -agonist caused a repression of 25 proteins in HIT+ß2 A compared to HIT, and an upregulation of 7 proteins compared to HIT. ß2 -Agonist repressed or even downregulated training-induced enrichment of pathways related to oxidative phosphorylation and glycogen metabolism, but upregulated pathways related to histone trimethylation and the nucleosome. Muscle contractile phenotype changed differently in HIT and HIT+ß2 A (P ≤ 0.001), with a fast-to-slow twitch transition in HIT and a slow-to-fast twitch transition in HIT+ß2 A. ß2 -Agonist attenuated training-induced enhancements in maximal oxygen consumption (P ≤ 0.01) and exercise performance (6.1 vs. 11.6%, P ≤ 0.05) in HIT+ß2 A compared to HIT. These findings indicate that daily ß2 -agonist treatment attenuates the beneficial effects of high intensity training on exercise performance and oxidative capacity, and causes remodelling of muscle proteome signature towards a fast-twitch phenotype.

Beta2 -adrenoceptor agonist salbutamol increases protein turnover rates and alters signalling in skeletal muscle after resistance exercise in young men.

KEY POINTS: Animal models have shown that beta2 -adrenoceptor stimulation increases protein synthesis and attenuates breakdown processes in skeletal muscle. Thus, the beta2 -adrenoceptor is a potential target in the treatment of disuse-, disease- and age-related muscle atrophy. In the present study, we show that a few days of oral treatment with the commonly prescribed beta2 -adrenoceptor agonist, salbutamol, increased skeletal muscle protein synthesis and breakdown during the first 5 h after resistance exercise in young men. Salbutamol also counteracted a negative net protein balance in skeletal muscle after resistance exercise. Changes in protein turnover rates induced by salbutamol were associated with protein kinase A-signalling, activation of Akt2 and modulation of mRNA levels of growth-regulating proteins in skeletal muscle. These findings indicate that protein turnover rates can be augmented by beta2 -adrenoceptor agonist treatment during recovery from resistance exercise in humans. ABSTRACT: The effect of beta2 -adrenoceptor stimulation on skeletal muscle protein turnover and intracellular signalling is insufficiently explored in humans, particularly in association with exercise. In a randomized, placebo-controlled, cross-over study investigating 12 trained men, the effects of beta2 -agonist (6 × 4 mg oral salbutamol) on protein turnover rates, intracellular signalling and mRNA response in skeletal muscle were investigated 0.5-5 h after quadriceps resistance exercise. Each trial was preceded by a 4-day lead-in treatment period. Leg protein turnover rates were assessed by infusion of [13 C6 ]-phenylalanine and sampling of arterial and venous blood, as well as vastus lateralis muscle biopsies 0.5 and 5 h after exercise. Furthermore, myofibrillar fractional synthesis rate, intracellular signalling and mRNA response were measured in muscle biopsies. The mean (95% confidence interval) myofibrillar fractional synthesis rate was higher for salbutamol than placebo [0.079 (95% CI, 0.064 to 0.093) vs. 0.066 (95% CI, 0.056 to 0.075%) × h-1 ] (P < 0.05). Mean net leg phenylalanine balance 0.5-5 h after exercise was higher for salbutamol than placebo [3.6 (95% CI, 1.0 to 6.2 nmol) × min-1  × 100 gLeg Lean Mass-1 ] (P < 0.01). Phosphorylation of Akt2, cAMP response element binding protein and PKA substrate 0.5 and 5 h after exercise, as well as phosphorylation of eEF2 5 h after exercise, was higher (P < 0.05) for salbutamol than placebo. Calpain-1, Forkhead box protein O1, myostatin and Smad3 mRNA content was higher (P < 0.01) for salbutamol than placebo 0.5 h after exercise, as well as Forkhead box protein O1 and myostatin mRNA content 5 h after exercise, whereas ActivinRIIB mRNA content was lower (P < 0.01) for salbutamol 5 h after exercise. These observations suggest that beta2 -agonist increases protein turnover rates in skeletal muscle after resistance exercise in humans, with concomitant cAMP/PKA and Akt2 signalling, as well as modulation of mRNA response of growth-regulating proteins.

Citrus bioflavonoids dipeptidyl peptidase-4 inhibition compared with gliptin antidiabetic medications.

This study compared dipeptidyl peptidase-4 (DPP-4) inhibitory activity of citrus bioflavonoid nutraceuticals compared with three gliptins. Citrus bioflavonoid standards and three commercially available citrus bioflavonoid supplements (Thompson's Super Bioflavonoid Complex®(SB), Ethical Nutrients Bioflavonoids Plus Vitamin C®(EN), and Country Life Citrus Bioflavonoids and Rutin®(CB)) were considered in this study. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis was undertaken to identify and quantitate the citrus bioflavonoids present in each supplement. The DPP-4 inhibitory activity was determined by fluorometric assay. All of the tested individual citrus flavonoids demonstrated DPP-4 inhibitory activity, with IC50 values ranging from 485 µM (rutin) to 5700 µM (hesperitin and eriodictyol). Similarly, the flavonoid supplements had IC50 values of 16.9 mg/mL (EN), 3.44 mg/mL (SB) and 2.72 mg/mL (CB). These values compare with gliptin IC50 values of 0.684 µM (sitagliptin), 0.707 µM (saxagliptin) and 2.286 µM (vildagliptin). The supplement flavonoid content varied from 11.98% (CB) to 5.26% (EN) and 14.51% (SB) of tablet mass, corresponding to daily flavonoid doses of around 300, 150 and 400 mg, respectively, with CB and SB containing rutin at levels of 7.0% and 7.5% of tablet mass, respectively. While our data demonstrated that citrus bioflavonoid based supplements do possess DPP-4 inhibitory activity, they are several orders of magnitude less potent than gliptins. Further studies using higher concentrations of citrus bioflavonoids, as well as investigations into antioxidant properties which may add additional benefit are warranted.

Peripheral treatment with enoxaparin exacerbates amyloid plaque pathology in Tg2576 mice.

Alzheimer's disease (AD) is a complex, progressive neurological disorder characterized by the formation of extracellular amyloid plaques composed of ß-amyloid protein (Aß), the key component in pathogenesis of AD. Peripheral administration of enoxaparin (ENO) reportedly reduces the level of Aß and the amyloid plaques in the cortex of amyloid precursor protein (APP) transgenic mice. However, the exact mechanism of these effects is unclear. Our previous studies indicated that ENO can inhibit APP processing to Aß in primary cortical cells from Tg2576 mice by downregulating BACE1 levels. This study examines whether ENO-induced reduction of amyloid load is due to the decreased APP processing to Aß in Tg2576 mice. Surprisingly, our results indicated that ENO significantly increases the Aß42/Aß40 ratio in cortex and enhances the amyloid plaque load in both cortex and hippocampus, although overall APP processing was not influenced by ENO. Moreover, ENO stimulated the aggregation of both Aß40 and Aß42 in vitro. Although ENO has been reported to improve cognition in vivo and has potential as a therapeutic agent for AD, the results from our study suggest that ENO can exacerbate the amyloid pathology, and the strategy of using ENO for the treatment of AD may require further assessment. © 2016 Wiley Periodicals, Inc.

Bronchopulmonary pharmacokinetics of (R)-salbutamol and (S)-salbutamol enantiomers in pulmonary epithelial lining fluid and lung tissue of horses.

AIMS: Salbutamol is usually administered as a racemic mixture but little is known about the enantioselectivity of salbutamol pharmacokinetics in the lung. This study was designed to investigate enantiomer concentrations in lung tissue after inhaled dosing. METHODS: Horses (n = 12) received racemic salbutamol 1000 µg via inhalation. Enantioselective ultra performance liquid chromatography-tandem mass spectrometry was used to determine salbutamol concentrations in pulmonary epithelial lining fluid (PELF) sampled 2, 5, 10 and 15 min after administration, in central lung (endoscopic bronchial biopsy) and peripheral lung (percutaneous pulmonary biopsy) tissues (at 20 and 25 min respectively), and in plasma samples. RESULTS: Mean ± 95% confidence interval (CI) yield of PELF was 57 ± 10 mg. Initial mean ± 95%CI (R)- and (S)-salbutamol PELF concentrations were 389 ± 189 ng g-1 and 378 ± 177 ng g-1 respectively, and both reduced approximately 50% by 15 min. Mean ± 95%CI central lung levels of drug were higher than peripheral lung tissue for both (R)-salbutamol (875 ± 945 vs. 49.5 ± 12 ng g-1 ) and (S)-salbutamol (877 ± 955 vs. 50.9 ± 12 ng g-1 ) respectively. There was no evidence of enantioselectivity in PELF or central lung but minor (~2%) enantioselectivity was observed in the peripheral lung. Enantioselectivity was clearly evident in plasma with (S):(R) ratio of 1.25 and 1.14 for both area under the concentration-time curve (0-25 min) and Cmax respectively. CONCLUSIONS: PELF sampling in horses offers sufficient yield allowing direct detection of drug and, combined with tissue sampling, is a valuable model to investigate bronchopulmonary pharmacokinetics. Salbutamol did not demonstrate enantioselectivity in PELF or central lung tissue uptake following acute dosing, however, enantioselective plasma concentrations were demonstrated, with minor enantioselectivity in the peripheral lung.

Terbutaline: level the playing field for inhaled ß2-agonists by introducing a dosing and urine threshold.

Terbutaline, a short-acting ß2-agonist similar to salbutamol, is widely used in Europe in the treatment of asthma and exercise-induced bronchoconstriction. Unlike salbutamol, terbutaline requires therapeutic use exemption (TUE) for therapeutic inhaled use in competitive sport. There is now compelling evidence that supratherapeutic use of terbutaline is performance enhancing, via oral dosing and inhalation. It is likely that the ergogenic effects of terbutaline are class specific for all ß2-agonists. The World Anti-Doping Agency (WADA) has introduced dosing and urine threshold and decision limits for other common ß2-agonists. This allows athletes to use these drugs for therapeutic purposes while minimising the potential for doping and administrative burden of TUEs. However, no such threshold limits currently exist for terbutaline. For terbutaline, athletes can be granted a TUE, then administer the drug via inhalation at supratherapeutic doses with impunity. The introduction of threshold dosing and urine limits for terbutaline should be a high priority, given the drug's demonstrated ergogenic effects.

Reliability of Haemophilus influenzae biofilm measurement via static method, and determinants of in vitro biofilm production.

Information is lacking regarding the precision of microtitre plate (MTP) assays used to measure biofilm. This study investigated the precision of an MTP assay to measure biofilm production by nontypeable Haemophilus influenzae (NTHi) and the effects of frozen storage and inoculation technique on biofilm production. The density of bacterial final growth was determined by absorbance after 18-20 h incubation, and biofilm production was then measured by absorbance after crystal violet staining. Biofilm formation was categorised as high and low for each strain. For the high biofilm producing strains of NTHi, interday reproducibility of NTHi biofilm formation measured using the MTP assay was excellent and met the acceptance criteria, but higher variability was observed in low biofilm producers. Method of inoculum preparation was a determinant of biofilm formation with inoculum prepared directly from solid media showing increased biofilm production for at least one of the high producing strains. In general, storage of NTHi cultures at -80 °C for up to 48 weeks did not have any major effect on their ability to produce biofilm.

Relationship between clinical site of isolation and ability to form biofilms in vitro in nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen associated with a range of infections, including various lower respiratory infections, otitis media, and conjunctivitis. There is some debate as to whether or not NTHi produces biofilms and, if so, whether or not this is relevant to pathogenesis. Although many studies have examined the association between in vitro biofilm formation and isolates from a specific infection type, few have made comparisons from isolates from a broad range of isolates grouped by clinical source. In our study 50 NTHi from different clinical sources, otitis media, conjunctivitis, lower respiratory tract infections in both cystic fibrosis and non-cystic fibrosis patients, and nasopharyngeal carriage, plus 10 nasopharyngeal isolates of the commensal Haemophilus haemolyticus were tested for the ability to form biofilm by using a static microtitre plate crystal violet assay. A high degree of variation in biofilm forming ability was observed across all isolates, with no statistically significant differences observed between the groups, with the exception of the isolates from conjunctivitis. These isolates had uniformly lower biofilm forming ability compared with isolates from the other groups (p < 0.005).

Relationship between cotinine and trans-3'-hydroxycotinine glucuronidation and the nicotine metabolite ratio in Caucasian smokers.

CONTEXT: Nicotine metabolite ratio (NMR) is linked with smoking behaviour and quitting, but little is known about the importance of UDP-glucuronosyltransferase metabolism phenotype on NMR. OBJECTIVE: To investigate the relationship between NMR and UDP-glucuronosyltransferase phenotype in a Caucasian population. METHODS: Urine samples from 260 smokers were measured by UPLC-MS/MS for cotinine (COT), 3'-hydroxycotinine, COT-glucuronide and 3'-hydroxycotinine-glucuronide. UDP-glucuronosyltransferase phenotype was assessed using the proportion of metabolites present as the glucuronide. RESULTS: A correlation was found between NMR and UDP-glucuronosyltransferase phenotype for COT (males and females) and 3'-HC in females (p < 0.0001). CONCLUSION: NMR is clearly influenced by UDP-glucuronosyltransferase phenotype for COT in Caucasians.

Proteoglycans in the central nervous system: role in development, neural repair, and Alzheimer's disease.

Proteoglycans (PGs) are major components of the cell surface and extracellular matrix and play critical roles in development and maintenance of the central nervous system (CNS). PGs are a family of proteins, all of which contain a core protein to which glycosaminoglycan side chains are covalently attached. PGs possess diverse physiological roles, particularly in neural development, and are also implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD). The main functions of PGs in the CNS are reviewed as are the roles of PGs in brain injury and in the development or treatment of AD.

Size and sulfation are critical for the effect of heparin on APP processing and Aß production.

Alzheimer's disease is associated with abnormal accumulation of Aß, which is produced from the ß-amyloid precursor protein (APP) by the ß-site APP-cleaving enzyme (BACE1) and γ-secretase. Our previous studies showed that heparin can decrease APP processing by decreasing the levels of BACE1 and ADAM10. In this study, we examined the effects of glycosaminoglycans (GAGs) on APP processing and Aß production with the aim of understanding the specificity of the effects. Various GAG analogs were incubated with primary cortical cells derived from APP (SW)Tg2576 mice and the level of APP, proteolytic products of APP and APP-cleavage enzymes were measured. The effect of GAGs on APP processing was both size- and sulfation-dependent. 6-O-Sulfation was important for the effect on APP processing as heparin lacking 6-O sulfate were less potent than native heparin. However, deletion of carboxyl groups on heparin had no significant effect on APP processing. Our studies suggest that there is structural specificity to the effect of GAGs on APP processing and that certain GAGs have a greater effect on Aß production than others. This suggests that it might be possible to alter the structure of GAGs to achieve more specific inhibitors of APP processing that can cross the blood-brain barrier.

The salmeterol anomaly and the need for a urine threshold.

Salmeterol is a long acting beta2-agonist (LABA) used widely for the treatment of airways disease. There is evidence that beta2-agonists, including salmeterol, have the potential for performance enhancing effects when delivered at supratherapeutic doses. For this reason, all beta2-agonists are currently on the Prohibited List issued by the World Anti-Doping Agency (WADA), regardless of dosing route with some exemptions for inhaled salbutamol, formoterol, and salmeterol when used at therapeutic inhaled doses. For 2020, salmeterol use is permitted up to a therapeutic dosing threshold of 200 µg daily, but unlike salbutamol and formoterol, there is an anomaly; currently there is no urine threshold to control for supratherapeutic dosing beyond this dosing threshold. Salmeterol, however, is reportable as an adverse analytical finding (AAF) at levels above 10 ng/mL. Complicating matters is that following inhalation, salmeterol parent drug is present at relatively low levels compared with other beta2-agonists due to rapid metabolism to the metabolite, alpha-hydroxysalmeterol, which is typically present at higher levels than the parent drug. Moreover, peak parent drug levels following permitted therapeutic dosing are below the minimum required performance level (MRPL) of 10 ng/mL for salmeterol (50% of the MRPL that analytical laboratories are required to meet for non-threshold beta2-agonists), hence the presence of salmeterol may be unreported. For consistency, a urine threshold should be introduced for salmeterol as a matter of priority, to balance the needs of athletes who use salmeterol therapeutically up to the agreed dosing threshold, with the need to control supratherapeutic dosing for doping intentions and athlete harm minimization.

Pharmacokinetic and pharmacodynamic effects of 2 registered omeprazole preparations and varying dose rates in horses.

BACKGROUND: Omeprazole preparations vary in bioavailability in horses. HYPOTHESIS/OBJECTIVES: To characterize the pharmacokinetics and pharmacodynamics of an existing enteric-coated oral omeprazole paste (REF) and a novel, in-feed, enteric-coated dry granule preparation (NOV). ANIMALS: Twelve Standardbred/Thoroughbred mares free from clinical disease. METHODS: A prospective, blinded randomized interventional study was trial, conducted in 3 parts: (a) bioavailability study, (b) dose titration study, and (c) comparative clinical pharmacodynamic study, each using a blocked crossover design. RESULTS: Consistent with the larger dose administered, Cmax (median, 1032 ng/mL; range, 576-1766) and AUC0-24 (median, 63.9 µg/mL*min; range, 42.4-152.4) were greater after single oral administration of NOV than REF (282.7 ng/mL; range, 94.8-390.2, and 319 23.8 µg/mL*min; range, 8.2-42.3, respectively; both P = .004). No differences were observed between products for absolute oral bioavailability (NOV 55% range, 15-88; REF 17% range, 10-77; P = .25). Treatment with both preparations was associated with reduced gastric squamous ulcer scores and increased pH of gastric fluid. Bioequivalence was demonstrated for pharmacodynamic measures with the exception of % time pH <4, despite differences in dose rate and subsequent plasma omeprazole concentrations. CONCLUSIONS AND CLINICAL IMPORTANCE: The findings of this study indicate that the NOV product would be a suitable alternative to the reference product, and confirm that plasma concentrations of omeprazole and omeprazole dose do not predict drug pharmacodynamics in horses.

An Abductive Inference Approach to Assess the Performance-Enhancing Effects of Drugs Included on the World Anti-Doping Agency Prohibited List.

Some have questioned the evidence for performance-enhancing effects of several substances included on the World Anti-Doping Agency's Prohibited List due to the divergent or inconclusive findings in randomized controlled trials (RCTs). However, inductive statistical inference based on RCTs-only may result in biased conclusions because of the scarcity of studies, inter-study heterogeneity, too few outcome events, or insufficient power. An abductive inference approach, where the body of evidence is evaluated beyond considerations of statistical significance, may serve as a tool to assess the plausibility of performance-enhancing effects of substances by also considering observations and facts not solely obtained from RCTs. Herein, we explored the applicability of an abductive inference approach as a tool to assess the performance-enhancing effects of substances included on the Prohibited List. We applied an abductive inference approach to make inferences on debated issues pertaining to the ergogenic effects of recombinant human erythropoietin (rHuEPO), beta2-agonists and anabolic androgenic steroids (AAS), and extended the approach to more controversial drug classes where RCTs are limited. We report that an abductive inference approach is a useful tool to assess the ergogenic effect of substances included on the Prohibited List-particularly for substances where inductive inference is inconclusive. Specifically, a systematic abductive inference approach can aid researchers in assessing the effects of doping substances, either by leading to suggestions of causal relationships or identifying the need for additional research.

Anabolic and lipolytic actions of beta2 -agonists in humans and antidoping challenges.

Inhaled beta2 -adrenoceptor agonists (beta2 -agonists) are among the most used substances in competitive sports. The 2020 Prohibited List issued by the World Anti-Doping Agency restricts use of all selective and non-selective beta2 -agonists in- and out- of competition with few exemptions. Formoterol, salbutamol, and salmeterol are allowed by inhalation within defined dosing limits. These restrictions are in place because supratherapeutic use of beta2 -agonist has the potential to be anabolic and to enhance performance, as well as due to potential side effects. Despite substantial documentation that beta2 -agonists exert anabolic and lipolytic actions, these actions are not widely recognized. Furthermore, a common misconception is that the inhaled route does not exert these effects. However, given the high relative systemic bioavailability via the inhaled route, inhalation at high doses can also exert anabolic and lipolytic actions. In this review, we highlight the anabolic and lipolytic actions beta2 -agonists can exert, regardless of the type of beta2 -agonist and the route of administration. The doses needed to provide such effects are also associated with adverse effects and would in most cases be detected in routine doping control. Notwithstanding, the beta2 -agonist regulations are associated with some challenges and given their ability to induce muscle growth and to enhance performance, it is important to continue developing effective detection strategies to prevent potential misuse of beta2 -agonists while allowing treatment of asthmatic subjects without causing adverse side effects or ergogenic actions.

Phytosterol supplements do not inhibit dipeptidyl peptidase-4.

BACKGROUND AND AIMS: Several commercially available phytosterol supplements are promoted for their cholesterol-lowering effects. However, limited information is available about their potential anti-hyperglycaemic effects. This study aimed to evaluate the dipeptidyl peptidase-4 (DPP-4) inhibitory effects of phytosterol supplements in silico and in vitro to determine their potential for anti-diabetic activity. METHODS: Docking studies were carried out in silico to evaluate the potential for interactions between three major phytosterol compounds (stigmasterol, ß-sitosterol, campesterol) and the DPP-4 enzyme, the enzyme that is inhibited by the anti-diabetic gliptins. Gas chromatography-tandem mass spectrometry (GC-MS/MS) was used to analyse three different supplements for phytosterol content. DPP-4 inhibitory activity was tested in vitro for these phytosterol supplements and two major phytosterol standards. RESULTS: In silico calculations predicted free binding energies for DPP-4 with the phytosterols to be: stigmasterol -8.78 kcal/mol; ß-sitosterol -8.70 kcal/mol; campesterol -8.40 kcal/mol. These binding energies indicated a potential for significant DPP-4 inhibition. However, these results were not supported by the in vitro studies. Stigmasterol and ß-sitosterol had an IC50 > 50 mg/ml (maximum tested concentration) and the Thompson's Cholesterol Manager® and Mega Strength Beta Sitosterol® supplements gave an IC50 > 100 mg/ml (maximum tested concentration). Blackmores Cholesterol Health® gave an IC50 value of 40 mg/ml which was attributed to ß-carotene content. CONCLUSIONS: Phytosterol supplements do not appear to offer any anti-diabetic activity potential via pathways that involve the inhibition of DPP-4.

Beta2 -adrenergic agonist clenbuterol increases energy expenditure and fat oxidation, and induces mTOR phosphorylation in skeletal muscle of young healthy men.

Clenbuterol is a beta2 -adrenoceptor agonist marketed as an asthma reliever but is not approved for human use in most countries due to concerns of adverse cardiac effects. Given its demonstrated hypertrophic and lipolytic actions in rodents, clenbuterol is one of the most widely abused doping substances amongt athletes and recreational body-builders seeking leanness. Herein, we examined the effect of clenbuterol ingestion on metabolic rate as well as skeletal muscle mammalian target of rapamycin (mTOR) phosphorylation and protein kinase A (PKA)-signaling in six young men. Before and 140 min after ingestion of 80 µg clenbuterol, resting metabolic rate and contractile function of the quadriceps muscle were measured, and blood samples as well as vastus lateralis muscle biopsies were collected. Clenbuterol increased resting energy expenditure by 21% (P < 0.001), and fat oxidation by 39% (P = 0.006), whereas carbohydrate oxidation was unchanged. Phosphorylation of mTORSer2448 and PKA substrates increased by 121% (P = 0.004) and 35% (P = 0.006), respectively, with clenbuterol. Maximal voluntary contraction torque decreased by 4% (P = 0.026) and the half-relaxation time shortened by 9% (P = 0.046), while voluntary activation, time to peak twitch, and peak twitch torque did not change significantly with clenbuterol. Glycogen content of the vastus lateralis muscle did not change with clenbuterol. Clenbuterol increased circulating levels of glucose (+30%; P < 0.001), lactate (+90%; P = 0.004), insulin (+130%; P = 0.009), and fatty acids (+180%; P = 0.001). Collectively, these findings indicate that clenbuterol is an efficient thermogenic substance that possibly also exerts muscle hypertrophic actions in humans. For these reasons, the restrictions imposed against clenbuterol in competitive sports seem warranted.

Effective reversed-phase ion pair high-performance liquid chromatography method for the separation and characterization of intact low-molecular-weight heparins.

A simple, selective, and efficient reversed-phase ion pair high-performance liquid chromatography (RPIP-HPLC) method was developed for the separation of various commercially available intact low-molecular-weight heparins (LMWHs). The developed method uses a C(18) column (150 x 4.6 mm) with diode array detection at 230 nm, flow rate at 1.0 ml/min, and a mobile phase containing acetonitrile/water (32:68%), tetrabutylammonium hydroxide (15 mM), and ammonium acetate (50 mM) at pH 7.0. The performance of this method was assessed in terms of selectivity, linearity, intra- and interday precision, and accuracy. The novel application of RPIP-HPLC with evaporative light scattering detection (ELSD) for the analysis of intact LMWHs was demonstrated. Intact LMWHs were analyzed with superior resolution and peak shape. Different chromatographic profiles were obtained for different LMWHs showing significant structural diversity. This method clearly showed chemical changes that occurred to LMWH under the stress condition. This method can be applied for the separation, identification, characterization, and pharmaceutical stability analysis of various LMWHs.