Results of the current study indicate that BHT can provide substantial protection against PUFA degradation in a DBS sample. Total HUFA composition in DBS remains stable in the presence of BHT for up to 21 days in open air although significantly lower levels are observed after 3 and 14 days of storage. Storage stability can be extended to at least 8 weeks when stored and capped in a test tube. These results are not in agreement with a previous study demonstrating that DBS samples wrapped and sealed in foil were unstable during a 2 month storage period. However, the specific BHT concentration used in this previous study is not reported, and as such 5.0 mg/mL of BHT in the present study may explain the improved stability. In addition, our samples were stored in test tubes or vacuum packed polypropylene bags that may provide a more protective seal than foil wrapping.
BHT mediated protection of PUFA is most likely due to free radical scavenging by BHT. The phenol group in BHT is thought to donate a proton to free radicals, thus neutralizing the free radicals and preventing them from accepting hydrogen protons from the methylene groups in PUFA and thereby preventing degradation. The results of the present study support BHT protection against PUFA loss as increasing concentrations of BHT on the chromatography paper demonstrates increased ability to prevent PUFA degradation. Additionally, storing DBS in sealable containers further prevents PUFA loss regardless of storage under an inert gas such as nitrogen.
During open air storage without BHT, omega-3 blood biomarkers responded differently that appear to be dependent on how each biomarker is calculated (Figure 1). Decreases in individual fatty acids from baseline were 23% for 18:2n-6 (data not shown), 33% for 18:3n-3 (data not shown), 47% for ARA, 55% for EPA and 61% for DHA (Table 1) that is in agreement with known kinetic rates of degradation. Omega-3 PUFA have a greater susceptibility for degradation as compared with omega-6 PUFA, as omega-3 PUFA generally contain more double bonds for a given chain length. The kinetic rates of degradation are 1.4 M-1 s-1 for 18:1n-9, 62 M-1 s-1 for 18:2n-6, 115 M-1 s-1 for 18:3n-3, 197 M-1 s-1 for ARA, 249 M-1 s-1 for EPA, and 334 M-1 s-1 for DHA[21, 22].
The significant drop in the % n-3 HUFA in total HUFA in DBS after 1 and 3 days in open air without BHT, with a subsequent return to baseline levels beginning at 7 days of storage is intriguing. This may reflect a higher degradation rate of susceptible omega-3 HUFA, with degradation of susceptible n-6 HUFA eventually “catching up” and resulting in a slight and subtle rebound of the % n-3 HUFA in total HUFA. In contrast, the sum of the % of EPA + DHA decreased rapidly and did not return under the same conditions. The % of EPA + DHA is calculated relative to the entire fatty acid pool including large amounts of fatty acids with relatively low degradation rates including palmitic acid (16:0), oleic acid (18:1n-9) and 18:2n-6.
Changes in the n-6/n-3 ratio support the hypothesis of differing degradation rates between omega-3 and omega-6 PUFA. The n-6/n-3 ratio demonstrates an inverse pattern as compared with the % n-3 HUFA in total HUFA with a rapid increase followed by a slight decrease as time passes. The n-6 PUFA with a slower degradation rate is in the numerator while the more rapidly degrading omega-3 PUFA is in the denominator. The differences in the n-6/n-3 ratio and % n-3 HUFA in total HUFA are likely due to the inclusion of 18:2n-6 in the n-6/n-3 ratio. The degradation rate of 18:2n-6 is much slower in comparison with 18:3n-3 and HUFA, and the amount of 18:2n-6 in whole blood is high (23.2 ± 0.4% of total fatty acids) as compared with the other PUFA (38.1 ± 0.8% of total fatty acids) based on baseline values. Although fatty acid peroxidation may still be occurring, the utilization of the % n-3 HUFA in total HUFA blood biomarker may provide a more useful and robust measure of omega-3 status during storage that can mask the effects of HUFA peroxidation and still provide an accurate omega-3 assessment. Our results suggest this may not be the case with the other omega-3 biomarkers assessed.
In the present study, purging with nitrogen did not provide additional protection against PUFA degradation during the 8-week storage period. It is unclear whether nitrogen purging would have provided additional benefits during a longer storage period. Previously, PUFA composition in O. cincta (springtail hexapod) has been maintained when saponification and transesterification was performed in nitrogen-filled headspace air. In addition, direct gassing of a dairy beverage enriched with 2% flaxseed oil with nitrogen protected against PUFA degradation, and nitrogen has also been used to slow oxidative degradation of perishable food products. In blood, nitrogen prevents fatty acid degradation in phospholipids for at least four weeks in plasma and less than four weeks in erythrocytes during storage at −20°C, and in erythrocytes for at least two years when stored at −80°C. Changes in HUFA composition during 8 weeks of nitrogen storage suggest that oxygen exposure may not be the only contributor towards peroxidation in blood samples during storage. In previous studies, erythrocyte exposure to air and pure oxygen in vitro for 48 hours at either 37, 26 or 4°C did not result in increased lipid peroxidation as measured by malondialdehyde formation. Plasma bubbled with nitrogen and stored at −20°C also did not prevent PUFA degradation. In healthy cells, approximately 3% of Hemoglobin(Hb)-(Iron)Fe2+ is converted to Hb-Fe3+ resulting in production of superoxide radicals (·O2)[28–30] that can generate lipid peroxyl radicals and lipid hydroperoxides. If the hydroperoxides are not efficiently removed, they can decompose to form more free radicals in the presence of iron that can further exacerbate oxidative damage. Thus, PUFA degradation in the present study may be a result of Fe2+-induced peroxidation mechanisms, and BHT has been shown to completely prevent this mechanism of PUFA peroxidation[31–33]. Other potential mechanisms of degradation may include light exposure, thermal degradation and humidity. Although humidity was not controlled for, all samples were stored in the absence of light and at a constant room temperature.
The present study has limitations. Our conclusions are limited to a single individual. A single, homogenous blood sample was used to enable extensive time point characterization of the PUFA degradation and restrict variation to the degradation process. Further assessment of the stability of PUFA in DBS samples need to be conducted on larger cohorts where the influence of other pro- and anti-oxidant blood materials on fatty acid degradation can be addressed. The current study however provides valuable information and timepoints and storage conditions to be examined. An additional limitation is that the baseline PUFA and HUFA levels were higher in study one as compared with baseline values in study two. Although blood samples for study one (different BHT levels) and study two (sealed storage comparisons) were taken from the same individual, they were collected at different time points. As such, comparing results between study one and study two must be made with caution as the potential for PUFA degradation may have been increased in study one. Total omega-3 PUFA measures of approximately 4% in study one and 5% in study two are higher than average DBS values determined in young Canadian adults, but similar to values determined in an elderly Canadian population. The higher omega-3 fatty acid composition of blood with the potential for increased peroxidation in the present study may therefore underestimate the PUFA stability of DBS samples from the general North American population.