Annual banned‐substance review: Analytical approaches in human sports drug testing

Several high‐profile revelations concerning anti‐doping rule violations over the past 12 months have outlined the importance of tackling prevailing challenges and reducing the limitations of the current anti‐doping system. At this time, the necessity to enhance, expand, and improve analytical test methods in response to the substances outlined in the World Anti‐Doping Agency's (WADA) Prohibited List represents an increasingly crucial task for modern sports drug‐testing programs. The ability to improve analytical testing methods often relies on the expedient application of novel information regarding superior target analytes for sports drug‐testing assays, drug elimination profiles, alternative test matrices, together with recent advances in instrumental developments. This annual banned‐substance review evaluates literature published between October 2016 and September 2017 offering an in‐depth evaluation of developments in these arenas and their potential application to substances reported in WADA's 2017 Prohibited List.     

Annual banned‐substance review: analytical approaches in human sports drug testing

The annual update of the list of prohibited substances and doping methods as issued by the World Anti‐Doping Agency (WADA) allows the implementation of most recent considerations of performance manipulation and emerging therapeutics into human sports doping control programmes. The annual banned‐substance review for human doping controls critically summarizes recent innovations in analytical approaches that support the efforts of convicting cheating athletes by improved or newly established methods that focus on known as well as newly outlawed substances and doping methods. In the current review, literature published between October 2008 and September 2009 reporting on new and/or enhanced procedures and techniques for doping analysis, as well as aspects relevant to the doping control arena, was considered to complement the 2009 annual banned‐substance review. Copyright © 2010 John Wiley & Sons, Ltd.     

The prognostic significance of T‐wave inversion according to ECG lead group during long‐term follow‐up in the general population

BackgroundInverted T waves in the electrocardiogram (ECG) have been associated with coronary heart disease (CHD) and mortality. The pathophysiology and prognostic significance of T‐wave inversion may differ between different anatomical lead groups, but scientific data related to this issue is scarce.MethodsA representative sample of Finnish subjects (n = 6,354) aged over 30 years underwent a health examination including a 12‐lead ECG in the Health 2000 survey. ECGs with T‐wave inversions were divided into three anatomical lead groups (anterior, lateral, and inferior) and were compared to ECGs with no pathological T‐wave inversions in multivariable‐adjusted Fine–Gray and Cox regression hazard models using CHD and mortality as endpoints.ResultsThe follow‐up for both CHD and mortality lasted approximately fifteen years (median value with interquartile ranges between 14.9 and 15.3). In multivariate‐adjusted models, anterior and lateral (but not inferior) T‐wave inversions associated with increased risk of CHD (HR: 2.37 [95% confidence interval 1.20–4.68] and 1.65 [1.27–2.15], respectively). In multivariable analyses, only lateral T‐wave inversions associated with increased risk of mortality in the entire study population (HR 1.51 [1.26–1.81]) as well as among individuals with no CHD at baseline (HR 1.59 [1.29–1.96]).ConclusionsThe prognostic information of inverted T waves differs between anatomical lead groups. T‐wave inversion in the anterior and lateral lead groups is independently associated with the risk of CHD, and lateral T‐wave inversion is also associated with increased risk of mortality. Inverted T wave in the inferior lead group proved to be a benign phenomenon.     

Targeting misuse of 2‐amino‐N‐ethyl‐1‐phenylbutane in urine samples: in vitro–in vivo correlation of metabolic profiles and development of LC‐TOF‐MS method

A phenyethylamine derivative, 2‐amino‐N‐ethyl‐1‐phenylbutane (2‐AEPB), has recently been detected in doping control and drugs‐of‐abuse samples, and identified as a non‐labelled ingredient in a dietary supplement. To facilitate efficient control of this substance we have studied the in vitro metabolic behaviour of 2‐AEPB with human liver preparation, compared these results with in vivo pathways in human, and finally propose an analytical strategy to target the potential misuse of 2‐AEPB for toxicological, forensic and doping control purposes. The major in vitro formed metabolites originated from desethylation (M1) and monohydroxylation (M2). A minor metabolite with hydroxylation/N‐oxidation was also observed (M3). In vitro‐in vivo correlation was studied in an excretion study with a single, oral dose of 2‐AEPB‐containing supplement. An unmodified substance was the most abundant target compound and detected until the last point of sample collection (72 h), and the detection of M1 (40 h) and M2 (27 h) demonstrated good correlation to in vitro results. In the study with authentic cases (n = 6), 2‐AEPB and M1 were mainly found in free urinary fraction, whereas higher inter‐individual variability was observed for M2. It was predominantly conjugated and already within this limited number of cases, the ratio between glucuronide‐ and sulpho‐conjugated fractions varied significantly. As a conclusion, hydrolysis is not mandatory in the routine sample preparation, and as the separation can be based on either gas chromatography or liquid chromatography, this study verifies that routine mass spectrometric detection methods targeted to amphetamine derivatives can be easily extended to control the misuse of 2‐AEPB. Copyright © 2014 John Wiley & Sons, Ltd.     

Structure-function relationships in the regulation of energy transfer between mitochondria and ATPases in cardiac cells

The present study discusses the role of structural organization of cardiac cells in determining the mechanisms of regulation of oxidative phosphorylation and interaction between mitochondria and ATPases. In permeabilized adult cardiomyocytes, the apparent K_m (Michaelis-Menten constant) for ADP in the regulation of respiration is far higher than in mitochondria isolated from the myocardium. Respiration of mitochondria in permeabilized cardiomyocytes is effectively activated by endogenous ADP produced by ATPases from exogenous ATP, and the activation of respiration is associated with a decrease in the apparent K_m for ATP in the regulation of ATPase activity compared with this parameter in the absence of oxidative phosphorylation. It has also been shown that a large fraction of the endogenous ADP stimulating respiration remains inaccessible for the exogenous ADP trapping system, consisting of pyruvate kinase and phosphoenolpyruvate, unless the mitochondrial structures are modified by controlled proteolysis. These data point to the endogenous cycling of adenine nucleotides between mitochondria and ATPases. Accordingly, the current hypothesis is that in cardiac cells, mitochondria and ATPases are compartmentalized into functional complexes (ie, intracellular energetic units [ICEUs]), which appear to represent a basic pattern of organization of energy metabolism in these cells. Within the ICEUs, the mitochondria and ATPases interact via different routes: creatine kinase-mediated phosphoryltransfer; adenylate kinase-mediated phosphoryltransfer; and direct ATP and ADP channelling. The function of ICEUs changes not only after selective proteolysis, but also during contraction of cardiomyocytes caused by an increase in cytosolic Ca²⁺ concentration up to micromolar levels. In these conditions, the apparent K_m for exogenous ADP and ATP in the regulation of respiration markedly decreases, and more ADP becomes available for the exogenous pyruvate kinase-phosphoenolpyruvate system, which indicates altered barrier functions of the ICEUs. Thus, structural changes transmitted from the contractile apparatus to mitochondria clearly participate in the regulation of mitochondrial function due to alterations in localized restriction of the diffusion of adenine nucleotides. The importance of strict structural organization in cardiac cells emerged drastically from experiments in which the regulation of mitochondrial respiration was assessed in a novel cardiac cell line, that is, beating and nonbeating HL-1 cells. In these cells, the mitochondrial arrangement is irregular and dynamic, whereas the sarcomeric structures are either absent (in nonbeating HL-1 cells) or only rarely present (in beating HL-1 cells). In parallel, the apparent K_m for exogenous ADP in the regulation of respiration was much lower than that in permeabilized primary cardiomyocytes, and trypsin treatment exerted no impact on the low K_m value for ADP, in contrast to adult cardiomyocytes where it caused a marked decrease in this parameter. The HL-1 cells were also characterized by the absence of direct exchange of adenine nucleotides. The results further support the concept that the ICEUs in adult cardiomyocytes are products of complex structural organization developed to create the most optimal conditions for effective energy transfer and feedback between mitochondria and ATPases.     

Comparison of management and 30-day mortality of acute myocardial infarction in men versus women in Estonia

OBJECTIVE: There is conflicting information about gender differences in clinical features, management and outcome after acute myocardial infarction (AMI). The objective of the study was to compare the baseline characteristics, management and 30-day mortality of AMI in men and women in Estonia. METHODS: This study included consecutive unselected patients from the Myocardial Infarction Registry (MIR) in Estonia, who were admitted to a university hospital between January 2001 and February 2002. Logistic regression analysis was used to estimate crude and adjusted odds ratios (OR) with 95 percent confidence intervals (95% CI). RESULTS: The study included 228 men and 167 women. Women were older than men (73.49 +/- 10.95 vs. 65.63 +/- 12.60, p < 0.000), and had more comorbidities. After age-adjustment, the higher prevalence of comorbidities, like diabetes (age-adjusted odds ratio [OR] 2.48, 95% confidence intervals [CI] 1.45-4.24), hypertension (OR 1.78, 95% CI 1.15-2.76) and history of congestive heart failure (OR 2.14, 95% CI 1.32-3.46) in women was preserved. Women were more frequently treated with diuretics (OR 2.68, 95% CI 1.69-4.25) and less frequently with statins (OR 0.61, 95% CI 0.39-0.96), after age-adjustment. Although thrombolytic therapy, coronary angiography and angioplasty were performed less frequently in women, these differences disappeared after age-adjustment. Female gender was not an independent predictor of 30-day mortality after AMI, crude OR was 1.39, 95% CI 0.80 to 2.41, adjustment for age and other covariates reduced OR to 0.98, 95% CI 0.44 to 2.20. CONCLUSIONS: Among AMI-patients, age but not gender is an important determinant of care and early mortality.     

Annual banned‐substance review: the Prohibited List 2008—analytical approaches in human sports drug testing

The list of prohibited substances and methods of doping issued by the World Anti‐Doping Agency is updated and modified annually based on most recent developments and scientific data. Compounds and methods are maintained, added, or removed from the list, or they are placed in so‐called monitoring programmes that have been established to obtain reliable data on the prevalence of particular substances and methods in‐ and/or out‐of‐competition. Consequently, doping control laboratories continuously update, modify and optimize existing screening and confirmation assays to ensure utmost comprehensiveness in detecting the prohibited and monitored substances as well as chemically and pharmacologically related analogs. The annual banned‐substance review for human sports drug testing critically summarizes recent innovations in analytical approaches supporting the detection of established and newly outlawed substances and methods of doping. Literature from January 2007 through September 2008 as indexed in Medline and Web of Science was screened and articles on detection methods for substances and methods of doping in humans were compiled according to the 2008 Prohibited List of the World Anti‐Doping Agency. Few new approaches were presented for individual doping agents and the majority of reports demonstrated new options for increasing the comprehensiveness of existing doping control assays. In addition, new techniques in separation and/or ionization of analytes complementary to commonly used procedures were described, which, so far, did not meet all requirements of sports drug testing. Copyright © 2009 John Wiley & Sons, Ltd.