Metherel Adam H, Stark Ken D
Department of Kinesiology, University of Waterloo, Waterloo, Canada.
Department of Kinesiology, University of Waterloo, Waterloo, Canada
J Nutr. 2015 Mar;145(3):654-60. doi: 10.3945/jn.114.203679. Epub 2015 Jan 7.
Fingertip prick whole blood collection on chromatography paper is amenable to high-throughput fatty acid (FA) profiling for large clinical and field studies. However, sample storage is problematic because highly unsaturated FAs (HUFAs) in erythrocytes rapidly degrade in samples stored at -20°C.
The aim of the current study was to determine the mechanism of HUFA degradation and to develop prevention protocols.
Free fatty acid (FFA) standards and whole blood reference material from a single participant were used to examine sample storage at -20°C for up to 90 d in triplicate. Iron chelation with deferoxamine (0-5000 μg), antioxidant protection with butylated hydroxytoluene (50 μg), cryopreservation with glycerol, and blood drying were examined using whole blood on chromatography strips. Biological replicate blood samples from additional participants (n = 6) with a range of ω-3 (n-3) HUFA concentrations were similarly assessed.
FFAs were relatively stable when stored on chromatography strips at -20°C. Glycerol treatment prevented HUFA degradation in whole blood reference material for 30 d (45 ± 0.4 to 46.8 ± 0.1, means ± SDs) compared to untreated saline controls (45.9 ± 1.0 to 6.8 ± 0.2). Pretreatment of paper for blood spots with deferoxamine and drying blood before storage slowed, but not entirely prevented, HUFA degradation over 30 d to 22% and 19% below baseline, respectively, compared to 86-92% in the controls. Protection against HUFA degradation with blood drying and glycerol treatment was confirmed in the biological replicate study and confirmed by prevention of cell lysis.
HUFA degradation during storage at -20°C appears to be due to hemolysis and subsequent iron-initiated peroxidation. This degradation may be prevented by glycerol, iron chelation, and/or dried blood spotting. A more thorough understanding of methods to prevent degradation during storage is critical with increasing use of FA profiling in large clinical studies.
在色谱纸上进行指尖采血用于高通量脂肪酸（FA）分析，适用于大规模临床和现场研究。然而，样品储存存在问题，因为红细胞中的高度不饱和脂肪酸（HUFA）在-20°C储存的样品中会迅速降解。
本研究的目的是确定HUFA降解的机制并制定预防方案。
使用来自一名参与者的游离脂肪酸（FFA）标准品和全血参考物质，一式三份在-20°C下检查样品储存长达90天。使用色谱条上的全血，研究了去铁胺（0 - 5000μg）的铁螯合、丁基羟基甲苯（50μg）的抗氧化保护、甘油冷冻保存以及血液干燥。对另外6名具有一系列ω-3（n-3）HUFA浓度的参与者的生物重复血样进行了类似评估。
FFA在-20°C下储存在色谱条上时相对稳定。与未处理的生理盐水对照（45.9±1.0至6.8±0.2）相比，甘油处理可防止全血参考物质中的HUFA降解30天（45±0.4至46.8±0.1，均值±标准差）。用去铁胺预处理血斑纸并在储存前干燥血液，可减缓但不能完全防止HUFA在30天内降解，分别比基线低22%和19%，而对照组为86 - 92%。生物重复研究证实了血液干燥和甘油处理对HUFA降解的保护作用，并通过防止细胞裂解得到证实。
在-20°C储存期间，HUFA降解似乎是由于溶血以及随后的铁引发的过氧化作用。这种降解可以通过甘油、铁螯合和/或干血斑法来预防。随着在大型临床研究中越来越多地使用FA分析，更深入地了解储存期间防止降解的方法至关重要。
