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dynamic measurement of metabolic profiles from hemaPEN microsamples

by James Rudge, PhD, Technical Director, Neoteryx | 6 min read

An article by Marianne Fillet et al at University of Liège in Belgium was published in the May 2021 edition of Frontiers in Molecular Biosciences. It reported on a proof-of-concept fluxomic study using microsamples of blood collected with a hemaPEN® device. The paper is entitled “Blood Microsampling to Monitor Metabolic Profiles During Physical Exercise.” It describes the development and validation of 13 targeted metabolomic biomarkers previously studied in sports science from volumetrically collected dried blood samples. The study involved collecting capillary samples from volunteers before, during, and after exercise, which would have been more challenging with standard venous blood draws. Indeed, the group concluded that their study demonstrated that microsampling with hemaPEN® followed by targeted metabolomics had great potential as a tool for the follow-up of metabolic profiles.runner-iStock-1030927082Human Biochemistry and a Focus on ‘Well-Health’

Understanding the complexity of the biochemistry of life is becoming of greater importance as scientists continue to develop more targeted and sophisticated therapies. Moreover, understanding perturbations in biochemistry helps physicians to diagnose and treat disease prior to physiological changes as an approach to preventing disease from developing. Indeed, the focus on human biochemistry has catalyzed a trend toward more research that focuses on ‘well-health.' It is hoped that such a focus will improve the lives of many and reduce the burden on healthcare systems.

We know that certain biochemical pathways change dependent on what we eat and how much we exercise. In many areas of medicine, it is important to monitor the degree to which biochemical pathways are impacted by diet and exercise. For example, it is well known that creatinine levels increase due to exercise as muscle fibers are broken down. This biochemical interrelationship is critical in the field of nephrology, where creatinine levels (amongst other analytes) of patients with kidney disease or kidney grafts must be closely monitored. Nephrologists routinely monitor their patients’ creatinine levels to measure the glomerular filtration rate (GFR). Nephrologists rely on blood samples from their patients to track elevated basal levels of this biomarker, which can help to indicate if there is a problem with a kidney transplant, or if there is kidney damage. However, exercise can also lead to increased creatinine levels, which can cause false positives if not taken into account.

Another example of a common biomarker associated with exercise is lactic acid. Lactic acid is a metabolite that can build up due to over-exercise, leading to cramped, oxygen-starved muscles and so is important to monitor. Lactic acid is a metabolite that can build up due to over-exercise, leading to cramped, oxygen-starved muscles, so it is important to monitor.

Understanding Multiple Metabolic Processes and Pathways

Metabolic profiling (the profiling of multiple metabolic processes and pathways) allows scientists to further understand the chemistry of life. However, there are several challenges researchers face in this field. The first is the utilization of expensive analytical instrumentation such as LC-MS/MS. A second challenge that investigators face is how to deal with the massive amounts of data that these techniques generate. A further challenge is how to monitor the dynamic nature of all components of the metabolome. One solution is to employ fluxomics, a term that describes a number of different approaches to determine the rate of metabolic reactions, either through real-time monitoring (technically challenging) or through repeated and rapid blood sampling over a short time period (e.g., over several hours).

One drawback to using conventional venous blood collection for repeated sampling over time is the logistics. This method of sample collection requires cannulation and the assistance of a phlebotomist. The conventional phlebotomy approach is possible if all study subjects are able to undergo blood sampling in one location, and if rapid sampling is not a requirement. However, this approach becomes problematic when attempting to collect samples remotely for a study that requires participants to be active and to utilize rapid sampling in the field, such as during sport activities. Indeed, it was the notion of rapid sampling of capillary blood using a finger-stick method with the portable hemaPEN that appealed to Marianne Fillet et al as a convenient way to measure the metabolic profiles of athletes. Fillet et al designed and carried out a study that monitored targeted metabolomic biomarkers in 20 health participants while they were alternating between rounds of exercise and rest.

The research group chose to use hemaPEN in the study for a number of reasons. First, it collects 4 volumetrically precise DBS samples simultaneously from a single drop of blood and stores the 4 blood spots inside the “pen” on a disk. Second, the samples collected are biologically identical (homogenous) and not effected by DBS disk area bias when using traditional sub punching. Moreover, the researchers highlighted the point that the hemaPEN device can be used anywhere by anyone without requiring formal training.

Metabolic Profile Study Methods and Results

The following targeted metabolomics biomarkers were selected because many of these had been identified in previous studies as interesting biomarkers for monitoring exercise:

  • L-asparagine, choline chloride, creatine, creatinine, L-leucine, L-methionine, L-proline, taurine, L-valine, 2-hydroxybutanoic acid, L-lactic acid, malic acid, and 2-oxo-glutaric acid.
Volunteers (n=20) were recruited (aged 19-30 years) with no known health conditions as non-smokers who exercised > 1hr per week. They were also asked not to drink or eat in excess the day before the study.

  • Prior to exercise, capillary blood was collected using a finger-stick with a lancet and a hemaPEN; volunteers were then asked to warmup and run a fast 800 m dash, after which the finger-stick blood sample collection was repeated. They were then asked to run another 800 m, followed by another capillary blood sample collection. Finally, the volunteers were asked to rest for 30 minutes, followed by blood collection. This final step was repeated one more time.

  • In total, each volunteer was sampled 5 times.

  • The hemaPEN devices were allowed to dry for 2h and then stored at -20° C before testing.

  • Prior to LC-MS/MS analysis, one disk of blood spots was removed from the inside of each hemaPEN, extracted and processed through an SPE plate for sample clean up. The rest of the hemaPEN devices were stored.

  • The assay was validated to the following parameters and all analytes passed these criteria:

    • Stability: 5 months’ stability stored at -20 °C at a <15% variance from the original value. All passed except for 2-oxoglutaric acid (-35.1%), methionine (-56.5%), creatine (+17.6%), and taurine (+15.7%).

    • Response Function, Trueness, Precision, and Accuracy returned values well below 20%.

    • Matrix effect was found to be around 76.6% for all but asparagine (117%).

    • Carryover, which should not exceed 20% of the peak area, was less than 2.07%.

  • Biomarker level changes during the experiment:

    • Creatinine, choline, and taurine were significantly increased, with creatine showing no sign of decreasing after the final blood sample

    • Lactic acid, malic acid and 2-oxoglutaric and 2-OH butanoic acids had all increased during the duration of the experiment.

Study Authors’ Discussion

The study group commented on the sampling benefits of hemaPEN stating, “The hemaPEN® device offers many advantages compared to classical blood sampling. Indeed, this type of blood collection can be done by everyone and everywhere.” They further stated that the simultaneous collection and secure sampling was also of benefit.
  • They highlighted the potential of the hemaPEN device for testing athletes, which may also be of interest to the World Anti-Doping Agency (WADA) for anti-doping programs.

  • Long-term stability was suitable for most analytes tested.

  • Due to the small sample number (n=20), the researchers were only able to perform limited statistical tests and couldn’t look at other parameters, such as sex, age, ethnicity, etc.

  • Many of the observations matched similar reported studies on monitoring fitness, such as the observed elevated levels of creatinine and lactic acid.

  • For some analytes, such as a number of the amino acids, the duration of the test may not have been long enough to see reported declines in biomarkers.

  • Further work would need to be conducted to look at other areas, such as untargeted markers.

Neoteryx Thoughts and Comments

This proof-of-concept study using the hemaPEN demonstrates good concordance with data from known published metabolomics studies and all from just 2.74 µL of blood! The ease of simultaneous sample collection, coupled with integrated desiccant in a tamper-resistant device such as the hemaPEN is highly attractive for those designing fluxomic and related studies. However, one point to mention is that it is always important to bridge between capillary and venous blood as there is sometimes a difference between the matrices.

For example, Marshall et al (as reviewed in a previous blog) showed that testosterone levels in capillary blood samples were lower than in venous blood samples even though other steroid hormones tested in the study did not show such a decrease. Nevertheless, the use of microsampling in omics is beginning to show excellent utility and, due to the ease of self-sample collection, the barrier to obtaining blood for such studies is much reduced compared to traditional sample collection methods. This efficiency helps scientists to delve deeper into the multiome and develop the next-generation targeted biomarkers for a world hoping to adopt personalized medicine as the gold standard of clinical tests.      

This study was summarized for our readers by James Rudge, PhD, Neoteryx Technical Director. This is curated content. To learn more about the important research outlined in this blog, visit the original study paper published in Frontiers in Molecular Biosciences.

Image Credits: iStock, Neoteryx

You can access this microsampling report and other studies in our Technical Resource Library.

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Originally published Jun 13, 2022 9:30:00 AM, updated on November 15, 2022

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