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eP032: Measurement of Nicotinamide Adenine Dinucleotide (NAD+) from dried blood spot cards

      Introduction

      Within the human body, all cells, from our neurons to our skin, require an energy source for function.
      ATP is the cellular energy source and is generated by mitochondria through oxidative phosphorylation.
      Nicotinamide adenine dinucleotide (NAD+) serves as an essential cofactor in the generation of ATP by the mitochondria. Due to this role in energy metabolism, measurement of NAD+ can provide insight into overall health and well-being. It is also a helpful tool in identifying supplementation needs and monitoring effectiveness.
      Previously, we demonstrated that NAD+ could be effectively stabilized on a chemically treated DBS card. This novel method was validated and the normal range was found to be consistent with reported ranges from methods that measured directly from whole blood. We have since expanded this work to continue to establish the validity of this approach and to explore the potential for at home sampling.

      Methods

      Three different experiments were designed for this study. The first was a patient matched study to allow for direct comparison of NAD+ measurements from the DBS method and a generally accepted whole blood-based method offered by NADmed (Helsinki, Finland). Twelve donors participated in the study. A dried blood spot and venous drawn whole blood (EDTA) sample was collected from each donor. DBS samples were analyzed within 48 h of collection. Whole blood samples were placed at ≤ − 80 °C immediately following collection and shipped on dry ice for analysis.
      Next, dried blood spot samples were collected from six individuals. Samples were analyzed after drying and then stored at room temperature (15 – 30 °C) and ≤ − 20 °C. NAD+ measurements were taken at various timepoints to establish stability. All measurements were compared back to the t=0 values.
      Finally, efforts have been made to refine the chemical treatment of DBS cards to determine if the stability of NAD+ could be further improved. DBS samples were collected from 11 donors on two sets of treated cards. The first set contained the original treatment and the second was an optimized coating. NAD+ measurements from each card type were compared.

      Results

      For the novel dried blood spot method, the average measured NAD+ concentration from the 12 donors was 24.81 μM with a range spanning 19.89 – 29.30 μM. The existing NADmed method resulted in an average of 25.85 μM with a range of 20.36 – 32.13 μM. The average difference between the two methods was 4.9%.
      The DBS stability study revealed that when stored at room temperature, 83% of NAD+ measurements taken after 7 days of storage were still consistent with the initial result. Storing at ≤ − 20 °C was found to significantly increase stability to 6 months of storage.
      The original card treatment resulted in an average NAD+ concentration of 27.74 μM across the 11 donors, with a range of 24.14 – 32.19 μM. For the optimized treatment, the average measurement was 34.08 μM with a range of 29.16 – 39.42 μM. In all cases, a higher NAD+ measurement was obtained from the optimized coating compared with the original (average increase was 18.5%).

      Conclusion

      The results of this study confirmed that NAD+ measurements from DBS are comparable to a previously validated whole blood method. The chemically treated DBS cards provide acceptable stability to allow for at home sampling, reasonable shipping conditions, and potential for sample batching within the laboratory. Optimization of the chemical coating further stabilizes NAD+ within the DBS card and thereby improves the robustness and quality of the assay.