Butylated hydroxytoluene can protect polyunsaturated fatty acids in dried blood spots from degradation for up to 8 weeks at room temperature

Abstract

BackgroundDried blood spots (DBS) from fingertip prick blood can enable high throughput fatty acid profiling but may be prone to lipid peroxidation during storage. The use of butylated hydroxytoluene (BHT) on chromatography paper can prevent polyunsaturated fatty acid (PUFA) loss but examinations on the length of storage times possible are not comprehensive.MethodIn the first study, venous whole blood was saturated on paper strips pre-soaked with 0, 2.5 or 5.0 mg/mL BHT and exposed to air for up to 28 days. In a second study, the effect of sealing DBS on 5.0 mg/mL BHT-soaked chromatography strips in capped test tubes or vacuum sealed polypropylene bags with and without nitrogen purging was examined over eight weeks. The fatty acid composition of the DBS were determined by gas chromatography and the effect of sample storage on omega-3 biomarkers were examined.ResultsPUFA and omega-3 biomarkers in DBS stored without BHT were dramatically reduced by day 3. In general, BHT delayed decreases in eicosapentaenoic + docosahexaenoic acid from baseline (3.2 ± 0.2 wt%) to 28 days (2.6 ± 0.03 wt%) of storage. In the % n-3 highly unsaturated fatty acids (HUFA) in total HUFA biomarker, BHT was more effective at preventing changes, particularly with 5.0 mg/mL BHT where no differences were detected up to 28 days. Sealed storage with BHT tended to increase the stability of the PUFA in DBS and nitrogen purging did not appear to provide additional benefits. The % n-3 HUFA in total HUFA biomarker also appeared to be more stable in the sealed storage study.ConclusionsThe storage of DBS in sealed containers with BHT may prevent PUFA degradation for up to 8 weeks. The % n-3 HUFA in total HUFA biomarker appears to provide a more consistent assessment of omega-3 status throughout storage as compared with other omega-3 blood biomarkers.

A method for long term stabilisation of long chain polyunsaturated fatty acids in dried blood spots and its clinical application

“…Furthermore, we chose to conduct many of our experiments on blood from an individual who was a long time consumer of high dose fish oil to deliberately subject our methodology to the magnification of having high blood levels (>15%) of n-3 LCPUFA. This is contrast to other reports [11,14] where the blood n-3 LCPUFA levels are only ~5% of the total fatty acids, such that small decreases in n-3 LCPUFA would be hard to detect.…”

“…Many of the reports claiming stable DBS methodology in profiling fatty acids are flawed by the use of inappropriate controls or using bloods with low levels of n-3 LCPUFA so that small changes would not have been detected [7,8,11,14,15]. Some studies [7,11,15] have taken as their zero time point a sample of blood that has been recently spotted onto filter paper and dried in air, a process we have shown here to result in significant losses in n-3 LCPUFA even in the presence of an antioxidant (Table 1).…”

“…Some studies [7,11,15] have taken as their zero time point a sample of blood that has been recently spotted onto filter paper and dried in air, a process we have shown here to result in significant losses in n-3 LCPUFA even in the presence of an antioxidant (Table 1). Two studies [8,14] claimed that their baseline blood spot samples were processed immediately at time zero, however, the actual air drying times were not indicated or controlled for. It is important to emphasise that our study used the fatty acid values from direct transesterification of 50µl of whole blood (that is, blood added directly to a tube containing transesterification reagent) immediately after phlebotomy as a baseline control for all of our studies, a process that ensures an accurate measure of original blood levels of n-3 LCPUFA [23].…”

“…In addition, the potential fatty acid contaminants released from the collection papers [10], and evidence of substantial oxidative losses of LCPUFA in DBS samples collected in this manner when stored in air [11] has led to concerns about the reliability of the method. Due to the high sensitivity of LCPUFA to oxidation, previous studies have taken the approach of processing the DBS samples immediately after blood collection [12,13], treating them with high levels of BHT [14] or storing them at -20°C to stabilise the LCPUFA [15]. However, none of these approaches fulfils the criteria for them to be considered as a stable and convenient clinical assay for large population studies, clinical trials or for pathology services.…”

Fast Transmethylation of Total Lipids in Dried Blood by Microwave Irradiation and its Application to a Population Study

“…Minimal changes in PUFA wt% were observed when samples were at room temperature for 5 days [21] or after two weeks [5]. The instabilities of fatty acid compositions in dried blood increased during long-term storage of up to 6 months, as reported in several studies [8, 10, 30]. A recent study on the long-term stabilities of fatty acids reported that pre-conditioning the filter paper with a higher concentration of BHT solution (5 mg/mL) could effectively maintain the n-3 % HUFA of dried blood, when stored at room temperature for 28 days [30].…”