Consent, capacity and compliance in concussion management: cave ergo medicus (let the doctor beware).

While the acute effects of concussion and mild traumatic brain injury (TBI) are well understood, the certainty in the medical literature regarding the long-term outcomes of sports-related concussion is limited. Long-term deficits that may result from single, repeated concussions, and possibly subconcussive impacts, include cognitive dysfunction, depression and executive dysfunction. Perhaps most troublingly, repetitive head impacts have been linked to neurodegenerative diseases, including chronic traumatic encephalopathy (CTE), although the precise risk of long-term consequences remains unknown. CTE represents a distinct tauopathy with an unknown incidence in athletic populations; however, a cause and effect relationship has not yet been demonstrated between CTE and concussions or between CTE and exposure to contact sports, as no prospective longitudinal studies have been performed to address that question. Studies of high-school sports exposure and long-term outcomes have not demonstrated consistent findings.Medical advice regarding return to play and the risk of acute and/or long-term consequences is therefore problematic. It is important that the individual's right to make their own choices regarding their health is respected. Team, coach, parental, peer or financial pressures should not influence this decision. The choice to return to play after a concussion or mild TBI injury is the athlete's decision once they have (1) recovered from their injury and have the legal capacity to make an informed decision; (2) been medically assessed and (3) been informed of any possible long-term risks in a language that they can understand.Given the current lack of certainty in relation to long-term outcomes from concussion, is it possible to provide a framework to inform players of current evidence, as part of a consent process, even if the information upon which the decision to return to sport is based remains uncertain and evolving?

Doxorubicin accumulation in individually electrophoresed organelles.

We report the doxorubicin content in individual organelles following their capillary electrophoretic separation and illustrate that chemical accumulation at the subcellular level is highly heterogeneous. In individual mitochondria from cultured human leukemia cells DOX amount is around 50 zmol, 2 orders of magnitude higher than expected from diffusion during drug treatment, and spans 2 orders of magnitude.

Subcellular metabolite profiles of the parent CCRF-CEM and the derived CEM/C2 cell lines after treatment with doxorubicin.

Micellar electrokinetic capillary chromatography with laser-induced fluorescence detection was used to detect the differences in doxorubicin metabolite accumulation in four subcellular fractions isolated from the CCRF-CEM and the CEM/C2 human leukemia cell lines. Five fluorescent metabolites and doxorubicin make up the metabolite profile of these cell lines upon treatment with 10 microM doxorubicin for 12 h, cell lysis, and fractionation by differential centrifugation. Based on the relative electrophoretic mobility of synthetic standards, we tentatively identify one metabolite as 7-deoxydoxorubicinone and suggest that doxorubicinone is not among those metabolites detected. Although the obvious difference between the derived cell line (CEM/C2) and the parent cell line (CCRF-CEM) is the decreased topoisomerase I activity in the former, the results presented here indicate that each cell line has a unique distribution of metabolites in each one of four subcellular fractions: nuclear-enriched, heavy-organelle-enriched, light-organelle-enriched, and cytoplasmic fractions.

Distribution of zeptomole-abundant doxorubicin metabolites in subcellular fractions by capillary electrophoresis with laser-induced fluorescence detection.

Doxorubicin (DOX) treatment of NS-1 mouse hybridoma cells results in the formation of zeptomole amounts of metabolites per cell that are difficult to determine by confocal microscopy or HPLC. The native fluorescence of DOX and its metabolites together with laser-induced fluorescence detection (HF) has previously been used to detect a maximum of four components. In this study, we use capillary electrophoresis with postcolumn LIF (CE-LIF) to separate and detect 12 components attributed to DOX metabolism, resulting from treatment of NS-1 cells with 25 microM DOX for 8 h. The so-called metabolites 8 and 10 have been identified as doxorubicinone (DOXone) and 7-deoxydoxorubicinone (7-deoxyDOXone), respectively, by comigration with the corresponding synthetic standard. Due to comigration of DOX with doxorubicinol (DOXone), the presence of DOXone had to be determined separately by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The rest of the metabolites remain unidentified and are referred to by their number assignment. In comparison with the whole cell lysate, fractionation by differential centrifugation results in a better separation resolution of metabolites due to reduced amounts of metabolites in each fraction. This approach was chosen to compare the distribution of 13 metabolites in three subcellular fractions that form a pellet at < 1,400 g, 1,400-14,000 g, and > 14, 000 g and that generically are enriched in nuclei, organelles (mitochondria and lysosomes), and cytosolic components, respectively. The most abundant metabolite, DOXone, was estimated to be 90 +/- 15, 18 +/- 2, and 60 +/- 12 amol/cell (n = 5) in the nuclear-enriched, organelle-enriched, and cytosole-enriched fractions, respectively. In contrast, the total amount of other metabolites in a given fraction varied from 0 to 1,300 zmol. 7-DeoxyDOXone is the only metabolite that was present at similar levels in the three fractions. Other salient observations are metabolites 3, 7, and 11 are not detectable in the nuclear-enriched, organelle-enriched, and cytosole-enriched fractions, respectively; metabolite 9 and DOXone are more abundant in the nuclear-enriched fraction than in the other two fractions. The observations presented here suggest that subcellular fractionation followed by CE-LIF could be a powerful diagnostic for monitoring drug distribution, which is highly relevant to DOX cytoxicity studies.

Detection of doxorubicin and metabolites in cell extracts and in single cells by capillary electrophoresis with laser-induced fluorescence detection.

Capillary electrophoresis with laser-induced fluorescence detection was used to separate and detect doxorubicin and at least five metabolites from NS-1 cells that were treated with 25 microM doxorubicin for 8 h. Using 10 mM borate, 10 mM sodium dodecyl sulfate (pH 9.3) as separation buffer, the 488-nm argon-ion laser line for fluorescence excitation, and a 635 +/- 27.5 nm bandpass filter for detection, the limit of detection (S/N=3) for doxorubicin is 61 +/- 13 zmol. This low limit of detection allows for the detection of a larger number of metabolites than previously reported. Two extraction procedures were performed: a bulk liquid-liquid extraction and an in-capillary single-cell lysis. While in the bulk liquid-liquid extraction procedure, recovery for doxorubicin range from 50 to 99%, in single cell analysis the recovery is expected to be complete. Furthermore performing lysis of a single cell inside the separation capillary prevents doxorubicin or metabolite loss or degradation during handling. Based on the bulk method the calculated metabolite abundance is in the sub-amol per cell range while it varies from 0.1 to 1.1 fmol per cell in single cell analysis confirming metabolite loss during handling. Each metabolite was found at a level less than 0.1% of the doxorubicin content in either method, suggesting a slow metabolism in the NS-1 cell system or effective removal of metabolites by the cell.