Simultaneous quantitation of oxidized and reduced glutathione via LC-MS/MS: An insight into the redox state of hematopoietic stem cells

Click here to read the full study in the journal: Free Radical Biology and Medicine

Cellular redox balance plays a significant role in the regulation of hematopoietic stem-progenitor cell (HSC/MPP) self-renewal and differentiation. Unregulated changes in cellular redox homeostasis are associated with the onset of most hematological disorders. However, accurate measurement of the redox state in stem cells is difficult because of the scarcity of HSC/MPPs. Glutathione (GSH) constitutes the most abundant pool of cellular antioxidants. Thus, GSH metabolism may play a critical role in hematological disease onset and progression.

A major limitation to studying GSH metabolism in HSC/MPPs has been the inability to measure quantitatively GSH concentrations in small numbers of HSC/MPPs. Current methods used to measure GSH levels not only rely on large numbers of cells, but also rely on the chemical/structural modification or enzymatic recycling of GSH and therefore are likely to measure only total glutathione content accurately.

 

 

Here, we describe the validation of a sensitive method used for the direct and simultaneous quantitation of both oxidized and reduced GSH vialiquid chromatography followed by tandem mass spectrometry (LC-MS/MS) in HSC/MPPs isolated from bone marrow. The lower limit of quantitation (LLOQ) was determined to be 5.0 ng/mL for GSH and 1.0 ng/mL for GSSG with lower limits of detection at 0.5 ng/mL for both glutathione species. Standard addition analysis utilizing mouse bone marrow shows that this method is both sensitive and accurate with reproducible analyte recovery.

This method combines a simple extraction with a platform for the high-throughput analysis, allows for efficient determination of GSH/GSSG concentrations within the HSC/MPP populations in mouse, chemotherapeutic treatment conditions within cell culture, and human normal/leukemia patient samples. The data implicate the importance of the modulation of GSH/GSSG redox couple in stem cells related diseases.

Aging May Impair Cells Ability to Respond to Exercise

If you’re an older adult, a 30-minute workout may not be as effective, even at the cellular level, as it was when you were younger. According to a new study, age may play a significant role in cell’s ability to respond to that activity.

The study, led by Dr. Tinna Traustadóttir of Northern Arizona University, was recently published in Free Radical Biology and Medicine, a journal of the Society for Redox Biology and Medicine (SFRBM).

In the study, a group of men age 18-30 were tested against a group of older men 55 years of age and older.  Study participants were generally healthy, non-smokers, who were not taking antioxidant supplements in excess of a multivitamin, or any non-steroidal anti-inflammatory drugs for two weeks leading up to their study visit.

The two groups cycled for 30-minutes, with blood being drawn at six time points to test cell function and antioxidant response. For this study, the exercise intensity was set relative to the individual’s maximal aerobic capacity as determined by a maximal oxygen consumption test during screening. It is known that older adults have a reduced aerobic capacity compared to young, thus this allowed the group to maintain the same relative workloads between young and old test groups.

“Through this study we were able to determine that an individual’s antioxidant response to exercise becomes suppressed with age,” said Dr. Traustadóttir an Associate Professor at Northern Arizona University and SFRBM Member. “Exercise is effective and critical for people of all ages, but this study shows that older adults do not achieve the same beneficial cellular responses as younger adults from a single bout of moderate exercise.”

The findings indicate a single session of submaximal aerobic exercise is sufficient to activate a important group of antioxidant genes at the whole cell level in both young and older adults. However, nuclear import of Nrf2, the regulator for this group of antioxidant genes, is impaired with aging. Nuclear import is required for Nrf2 to access the antioxidant gene targets. Together these data demonstrate for the first time the weakening of Nrf2 activity in response to exercise in older adults.

Traustadóttir’s ongoing research aims to identify molecular processes responsible for age-related cellular changes. By better understanding the molecular signals promoting beneficial effects of exercise, definitive recommendations could be made for improving the body’s reaction to oxidative stress, which could lower the risk for many chronic diseases.

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John Oliver Has It Right – Not All “Science” Is Created Equal

Post by: Peter Vitiello, PhD 

Last week, HBO’s satirical comedy newsman, John Oliver, addressed how scientific studies can be misconstrued by the media, transforming their original findings into entertainment solely suitable for morning show banter. In addressing how junk science becomes overhyped, the episode did a great job exposing data replication issues limiting research investments.

(Click Here to View HBO’s John Oliver Video Clip. Warning: Language may be inappropriate for some.)

Reproducibility became an important topic of discussion in 2012 when Amgen reported that they were unable to reproduce findings in 47 of 53 “landmark cancer papers” published in journals with an impact factor greater than five. Even when considering failure rates of pre-clinical studies, this 94% irreproducibility rate was shocking and served as the motivation for new standards. The US National Institute of Neurological Disorders and Stroke called for data reporting standards in regard to animal randomization, blinded assessment, sample-size estimation, and data handling and biological sex and reagent authentication have also been added to this list. However, there was also a clear delineation between how this rigor should be applied exploratory studies (early-stage observational tests) and more robust hypothesis-testing experiments.

In light of such issues, direct requests to access raw data and protocol details of published studies have increased. However, editors of the New England Journal of Medicine, which coincidentally has the highest retraction rate of any journal in the world, recently refereed to such scientists as “research parasites”. Casey Greene at the University of Pennsylvania Perelman School of Medicine used this opportunity to create the Research Parasite Award to recognize outstanding contributions to the rigorous secondary analysis of data (nominations are due by October 14). More transparent approaches for handling confirmatory and replicative studies performed by such “parasites” are being developed. Although Amgen’s 47 irreproducible studies remain anonymous, they recently released data on three failed studies through a new “Preclinical Reproducibility and Robustness” channel created by Faculty of 1000. Many other publishers are following suit as Elsevier recently announced a new “Invited Reproducibility Paper” as a new article type appearing in the data science journal, Information Systems.

In summary, I’m very excited to see greater data sharing and transparency through guidelines set by both funding agencies and publishers along with opportunities and respect for parasitic confirmatory studies. I hope that scientists embrace such expectations and do not use them as sole justification to dismiss creativity and novelty during peer review. My only fear is knowing that my harshest critics watch John Oliver and I’ll be expected to respond to these cynics at our next family gathering.

  1. Season 3: Episode 11. in Last Week Tonight with John Oliver (HBO, 2016).
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  3. Landis, S.C., et al. A call for transparent reporting to optimize the predictive value of preclinical research. Nature 490, 187-191 (2012).
  4. Oransky, I. & Marcus, A. For science to improve, let’s put the right prizes on offer. (STAT, 2016).
  5. Baker, M. Biotech giant publishes failures to confirm high-profile science. Nature 530, 141 (2016).
  6. Chirigati, F., Capone, R., Rampin, R., Freire, J. & Shasha, D. A collaborative approach to computational reproducibility. Information Systems (2016).