Antioxidant Mechanisms of Docosahexaenoic Acid (DHA): Research, Analysis, and Reflection – Blog 2

This summer, Dr. Upmacis and I have sought to understand the antioxidant potential of docosahexaenoic acid (DHA) by examining its interaction with oxygen.  Since a structurally similar polyunsaturated fatty acid known as eicosapentaenoic acid (EPA) has been shown to progressively oxidize in the presence of air, we expected DHA to behave in much the same way when subjected to the same circumstances.  In my last blog post, I described our use of mass spectrometry to test this hypothesis.  Dr. Upmacis and I dissolved DHA separately in water and ethanol, then left both samples exposed to air for a period of five days.  Each day, we processed both samples in the mass spectrometer several times in order to glean a consistent picture of each sample’s chemical constituents.  Our first three daily trials collected intensity data at 0.1 mass-to-charge (m/z) increments[1] ranging from 50 m/z to 650 m/z. We also conducted two additional daily trials, expanding our scope of measurement beyond 650 m/z in order to hunt for even larger molecules.  The final result was five days worth of detailed atomic mass spectra consisting of multiple trials per sample per day.

 

The configuration of our atomic mass spectra has raised some interesting questions and curiosities, which we have attempted to rationalize.  In our primary trials, we noted that the most prominent base peaks usually occurred at about 327.4 m/z and 328.4 m/z; we think these peaks correspond to the molar mass of the deprotonated and protonated forms of DHA respectively.  Similarly, we believe a hydrogen ion (proton) is responsible for single unit gaps between twin peaks that occur elsewhere in our spectra.  We also noted a rhythm of peaks occurring at regular intervals of 16 m/z beyond the main peak.  As the sample aged, the main peak declined, while the series peaks that followed grew in intensity.  This finding appears to be consistent with our hypothesis that DHA takes on one or more oxygen – an atom weighing 16 amu – when progressively exposed to air.  Our additional trials on extended m/z ranges revealed another fascinating discovery: the emergence of an additional prominent peak at 654.8 m/z.  We noted this peak to be precisely double the m/z of the main base peak but roughly half the intensity.  We suspect this phenomenon provides evidence for the dimerization of the initial DHA compound.  A rhythmic succession of peaks also followed the 654.8 m/z peak at regular intervals of 16 m/z (corresponding to the size of an oxygen atom) and at 32 m/z (corresponding to the size of a pair of oxygen atoms, which might join to the possible dimer).  Finally, we turned our attention back to earlier ranges of the spectra, where we found another rhythmic succession of peaks.  Curiously, this series actually preceded the 327.4 m/z main base peak, which initially puzzled us.  However, we now postulate that these peaks actually represent an “echo” of larger molecules that have become doubly charged.  The double charge sets the m/z denominator (z) = 2, rendering an m/z value corresponding to half the given molecule’s true size.

 

The majority of time since my last blog post has been spent analyzing and making sense of our empirical data.  Because we conducted five trials for each sample over five days, we amassed an enormous cache of data.   Furthermore, each trial reported m/z values in 0.1 increments, and in some trials, values ranged upwards of 1800 m/z.  This culminated in a grand yield of a quarter-million distinct data-points across the entire project!  To complicate matters, it was necessary to align the data so that m/z values from one day could be meaningfully compared with those of the next day.  Initially, I attempted this task manually, by placing tabular data sets side-by-side in Microsoft Excel and moving misaligned records into position.  Ultimately, I found this method impractical and tedious, and so I abandoned it in favor of an automated approach.  I therefore attempted to construct an Excel macro that would align the data automatically, consulting an external Excel expert to assist me with portions of Visual Basic software coding, programming syntax, and debugging.  After weeks of continued development and thorough testing, our macro evolved from a simple data-aligning routine to an intelligent, sophisticated program capable of retrieving whole datasets from multiple source files, initiating user-driven comparisons, and assembling graphical overlays so as to highlight changes occurring in our samples from one day to the next.

 

In order to more fully understand the detailed patterns and relationships inherent in the data, I am currently zooming into local areas of each trial’s graph and labeling each peak.  I am noticing that some day-to-day peaks grow, some decline, and others do not seem to conform to any recognizable pattern at all.  Nevertheless, I anticipate this careful examination of peaks under magnification will uncover even more numerical patterns within our atomic mass spectra.  We hope an understanding of these patterns will help unlock the mysteries behind the chemistry of DHA, particularly with respect to our original hypotheses.

 

Next week, Dr. Upmacis and I plan to simulate DHA’s antioxidant mechanisms as they realistically occur within the body.  We will observe the reactions between DHA and several nitric oxide (NO)-releasing compounds, including S-Nitroso-N-acetylpenicillamine (SNAP) and S-Nitrosoglutathione (GSNO).  NO is a radical species important in many physiological and pathological processes.  We expect DHA to scavenge NO and couple with NO’s unpaired electron.  If these tests are successful, we will be one step closer to painting a more complete picture of the antioxidant potential inherent in DHA.

 

This project has certainly presented its own set of challenges, but I felt rewarded by the satisfaction earned by overcoming them.  My first obstacle lay in the alignment of my datasets.  I felt overwhelmed by the prospect of tediously combing through tens of thousands of data records, one by one.  Moreover, I realized such a manual approach was vulnerable to human error.  I knew there had to be a more efficient method, so I began to envision a program that would automate the task.  This gave rise to my next challenge: designing and testing the Excel macro.  Many steps were taken to ensure the macro sorted the data correctly.  A new round of thorough testing accompanied each additional feature or level of complexity.  This process required a significant investment of time.  However, the result was well worth the effort because it yielded a flexible, self-sufficient program capable of sorting and analyzing data under varying conditions.  I am hopeful that this macro will also improve the repeatability of our experiment, should a future study necessitate a similar data analysis.  In this way, the investment of time and effort here will continue to pay off in the future.  Unfortunately, the amount of time spent in research and analysis initially left me feeling rushed to produce more results by summer’s end.  This brings me to my final challenge – the time constraints of this project.  Many studies often take many months or years to come to fruition, so I initially felt rushed to complete my work before the end of the initiative.  I’m now learning that the research and analysis process requires the time that proper diligence and care demand.  Therefore, it may be unrealistic to expect that ambitious research undertakings will be completed within a relatively short time frame.

 

That said, Dr. Upmacis and I will be starting a new project in the fall, but we will continue our work with the current one as well.  Once the numerical patterns behind our mass spectra peaks are more fully elucidated, we will assemble a cohesive narrative to explain the chemistry of DHA in terms of the changing relationships in our data set.  This story will form the backbone of our manuscript, which we may submit for publication.

 

This project has taught me much about the research process.  I’m thankful for the chance to work with Dr. Upmacis, a phenomenal mentor, who has guided me through the journey of the scientific method.  I also feel empowered by the knowledge and technique that I’ve acquired in the lab.  I learned how to use the mass spectrometer, an important instrument in analytical chemistry.  Moreover, I gained valuable skills in critically evaluating data and drawing meaningful conclusions.  Helping this study come together has been a rewarding experience and will continue to be one as we approach our goal.

 

In addition to learning the ropes of research, this project has also enhanced my sense of patience and resolve.  There is rarely immediate gratification in the worthwhile endeavors of life; one has to be in it for the long haul.  It’s easy to become discouraged or frustrated when long hours produce slow results.  The key to success, however, is to stay engaged in the process, to pace oneself, and to never give up.  Give your best effort with the tools available to you at present, stay enthusiastic, and success will take care of itself.  This is a lesson that I will carry on through my life’s ventures and professional pursuits.

 


[1] Assuming the mass spectrometer singularly charges component molecules, then z = 1 and “m/z” directly represents the atomic mass units (amu) of the molecule.  Some molecules, however, inevitably become doubly charged; in this case, z = 2 and “m/z” represents half the amu of the molecule.  Thus, for accuracy, we report our data in terms of m/z units, rather than amu.

Progress of Videogaming on Cognitive Abilities

From the limited data that we gathered from our research it appears that screen exposure impacts certain individuals differently, making some more susceptible to reduced ability of inhibition and attention. The data, however, was not confined to one effect. It was found that some participants who played videogames on a regular basis were still able to successfully perform the tests measuring their attention and inhibition after completing the videogame control whereas some scored the same as they had when measured before the control, others improved, and other declined. Participants who were less accustomed to screen exposure also had the same results: some scored higher after the electronic control whereas others scored a lower or an equal amount.
Time was an important factor. It may be that the 10 minutes each subject was given to complete each control was not long enough. The amount of time each subject was allotted could also have contributed negatively to our results because in the real world the average college student isn’t exposed to only 10 minutes of screen time.
Practice effects also appear to be influencing results. Some subjects had already been exposed to the STROOP Color and Word test through school and the internet, I also noted that majority of subjects scored higher on the STROOP the second time it was given to them after each control because they were more familiar with what it entailed and what was expected of them to achieve a higher score. For example, one subject in particular vocalized when he received the STROOP for the first time that he couldn’t read the words quickly in fear of making a lot of careless mistakes, however, when given the test for the then second time after the control he was able to read more quickly because now he knew what the test entailed and was no longer worried about making errors. This possibly shows that those who obtained a higher score on this test did so because they were familiar with it not because one control affected their cognition negatively or positively.
This project has given me insight on how to work with people from a researcher standpoint. When working with human subjects patient and persistence is highly essential. From scoping, to contacting, to reminding, to participating each step requires the researcher to make the subjects feel important by knowing that they are contributing to data that can have a profound impact of the community at large.

Where I am in the process…

When I initially applied to this research program, I had no idea how much work goes into simply getting research APPROVED. The process of submitting work to the IRB can come with an excruciatingly long waiting period, when the researchers have no idea whether to progress on, assuming their work will be approved, or to halt all investigations until there is a 100% assurance that their work is valid and ethical. In the time that it took for the IRB to approve our surveys, I started to feel anxious, a little annoyed that it would take so long, and honestly…I became a little lax in my research. Professor Molina and I had completed our surveys months ago, and only just distributed them to our demographic for feedback. We yielded sixty-one responses. I am very proud of those responses, and can now begin a more in-depth and involved research process. Up until now, I had felt a little lazy in my research, as I did not have much to work with besides what research has already been done on the topic. Not surprisingly, much work has been done on the sibling relationship of people with autism. Especially over the last decade or so when autism diagnosis has been on the rise – it means that even more siblings are effected. What makes our research unique is that we are working with college-age participants, unlike children or babies with whom most autism-sibling research is done. People with autism in college obviously have what it takes to be an integral member of the social community and who are on the precipice of starting their own lives. Their perspective on their sibling support is much deeper and stronger than that of children, whose relationships with their siblings is just beginning to develop.

After Professor Molina submit our survey to the IRB, he requested that I partake in and complete a three hour online course that teaches about the ways to protect human research participants. I completed the course, and am now fully certified and educated in the proper treatment of humans used in a research process. It is not just a matter of asking questions, observing and getting answers. The questions you ask must be ethical. You must inform the participants on ever facet of the research, and they must know that they can withdraw their participation at any time. There are many rules that go into being qualified to utilize humans in your research, and I had no idea of these things prior to this course.

We will now proceed to extrapolate from our responses data to put into charts. We will compare and contrast the responses, and see how we can generalize the information. Now that we have concrete data, we can really dig into finding out more about how effective and beneficial it is for people with autism to have siblings to support them. I know I will personally make up for the time that was lost in our research due to waiting for approval.

 

 

Autism and Siblings

At this time in our research, we have completed our primary questionnaires for both college-aged people with Autism, as well as people who are siblings of people with Autism in college. We are currently waiting for the IRB to approve our questions, so we can then proceed with distributing these questionnaires and compiling data.

As we do not have any solid data yet, I can only speculate on what sort of things we will find. I hope that our participants are candid and honest, as that will help us best in our research. This may be a very sensitive subject matter, and talking about personal experiences may pose a challenge to many participants, I think it can be very cathartic to talk about difficult things. Sometimes people with Autism do not have any one to talk to about their personal experiences, and their day-to-day life is very regimented and there is no time to reflect on their emotions or daily experiences. Our research will give people will Autism a chance to talk about and explore a topic they may not have a chance to discuss otherwise. What do their siblings mean to them? Have they ever recognized their siblings as life-long teachers? How do they think they would have been different without their sibling(s)?

I also think this process will let the siblings of people with Autism discover how truly instrumental they are, and will continue to be in their Autistic sibling’s life. It will allow them the opportunity to reflect on all they do for their sibling. I hope that they feel like they can open up. If both parties are honest in their answers, we can gather a lot of important data from their answers. I look forward to the IRB’s approval of our questionnaire so we can begin!

Identifying the Effectiveness of Sibling Support for Individual’s Diagnosed with Autism Spectrum Disorder

The purpose of this research is to identify and characterize the benefits of sibling support for people on the Autism spectrum. Our demographic focus is on college students, between the ages of 17-25, as siblings often play a huge role in support at this transitional juncture in a person’s life. Siblings of all ages offer people with Autism uniquely different, but key levels of support. This support can motivate them, teach them and push them forward while they acclimate to the college setting. We will outline and narrowly define our definition of support in our research. Through surveying people in college with Autism Spectrum Disorder, as well as siblings of people with Autism Spectrum Disorder, we intend to find specifically what sibling support does for people on the spectrum. Siblings are often the best role models, as their relationship is lifelong, and extends past parental relationships, and friendships, which can come and go. A sibling can teach you, simply by existing in your world. Siblings of people with Autism teach invaluable lessons, which often cannot be taught in school programs. They can teach a person with Autism things like how to forge meaningful relationships, how to handle stressful situations, and how to be a self-advocate, which are all examples of important lessons to know before college, or during college.

This research will be conducted through surveys, as well as a second component of personal, individual interviews. This research will shed light on how people with Autism interpret their sibling support, how siblings of people with Autism understand the support they offer, and maybe even how studying these important sibling relationships can offer insight on how best to cater teaching to people in the college setting with Autism Spectrum Disorder. We hope to give a voice to a demographic who are rarely discussed; the siblings of people with Autism, and to shed light on their meaningful relationships with their brother or sister with Autism.