The stability of blood fatty acids during storage and potential mechanisms of degradation: A review

Prostaglandins, Leukotrienes and Essential Fatty Acids 104 (2016) 33–43

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Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa

Review

The stability of blood fatty acids during storage and potential mechanisms of degradation: A review Adam H. Metherel, Ken D. Stark n University of Waterloo, 200 University Avenue, Waterloo, ON, Canada N2L 3G1

art ic l e i nf o

a b s t r a c t

Article history: Received 25 August 2015 Received in revised form 3 December 2015 Accepted 5 December 2015

Fatty acids in blood samples, particularly polyunsaturated fatty acids (PUFAs), are susceptible to degradation through peroxidation reactions during long-term storage. Storage of blood samples is necessary in almost all studies and is crucial for larger clinical studies and in field research settings where it is not plausible for analytical infrastructure. Despite this, PUFA stability during blood storage is often overlooked. This review introduces and discusses lipid peroxidation and popular strategies employed to prevent or minimize peroxidation reactions during fatty acid analysis. Further, an in-depth examination of fatty acid stability during storage of blood is discussed in detail for all blood fractions including plasma/serum, erythrocytes and whole blood stored both in cryovials and on chromatography paper before discussing the associated mechanisms of degradation during storage. To our knowledge this is the first review of its kind and will provide researchers with the necessary information to confidently store blood samples for fatty acid analysis. & 2015 Elsevier Ltd. All rights reserved.

Keywords: Fatty acid Polyunsaturated fatty acid Storage stability Peroxidation Blood

Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lipid peroxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Strategies to prevent fatty acid losses during storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fatty acid stability during storage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Plasma/serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Erythrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Whole blood in cryovials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Whole blood on chromatography paper. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Mechanisms of fatty acid losses during storage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Authorship. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

33 34 35 35 35 37 38 39 39 41 41 41 41

1. Introduction Abbreviations: FTP, fingertip prick; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; HUFA, highly unsaturated fatty acid; PUFA, polyunsaturated fatty acid; ARA, arachidonic acid; Hb, hemoglobin; BHT, butylated hydroxytoluene; PL, phospholipid; TAG, triacylglycerol; CE, cholesteryl ester; NEFA, non-esterified fatty acid; LNA, linoleic acid; PLA2, phospholipase A2; lyso-PC, lysophoshatidylcholine; TLE, total lipid extract; EDTA, ethylene diamineteraacetic acid; DBS, dried blood spots; Fe2 þ , ferrous iron; Fe3 þ , ferric iron n Correspondence to: Department of Kinesiology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1. Tel.: þ 519 888 4567x37738; fax: þ519 885 0470. E-mail address: [email protected] (K.D. Stark). http://dx.doi.org/10.1016/j.plefa.2015.12.003 0952-3278/& 2015 Elsevier Ltd. All rights reserved.

The use of omega-3 blood biomarkers in clinical studies has increased dramatically since 2004 with the proposal of the sum of the relative percentage of eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) in erythrocytes as a potential risk factor for coronary heart disease [1], and the development of rapid fingertip prick (FTP) capillary blood collection techniques for fatty acid profiling [2]. EPA þDHA in erythrocytes (also known as the “Omega-3 index”) and other fatty

34

A.H. Metherel, K.D. Stark / Prostaglandins, Leukotrienes and Essential Fatty Acids 104 (2016) 33–43

acid blood biomarkers continue to be used to assess cardiovascular disease risk [3], as initially proposed, but also for maternal and infant health [4,5] and dietary intake assessments [6,7]. Whole blood fatty acid assessments via fingertip prick collection have become common practice due to ease of collection, minimal invasiveness and amenability to high-throughput analytical techniques, however, translational equations to estimate erythrocyte EPA þDHA equivalents [8] may still be required for health risk assessments as patterns of incorporation of EPA and DHA in blood fractions are different [9]. Improved analytical throughput via FTP blood collection can increase the reliance on temporary blood storage, such as during clinical studies that collect thousands of samples and may require years of storage prior to analysis, and during field research. Ultracold storage infrastructure is often limited or unavailable, particularly in the latter scenario. In addition, both clinical and field research frequently requires shipment of samples from multiple locations to specialized fatty acid assessment laboratories, which adds additional questions regarding storage stability. Although many study protocols require that blood samples are stored for long periods of time, it is often overlooked that under certain storage conditions fatty acids may be highly susceptible to degradation through lipid peroxidation. Making fatty acid assessments and storage stability more difficult to understand and interpret is that analytical techniques employed for fatty acid determinations are variable and depend largely on the sample of interest. Plasma [10], erythrocytes [11], whole blood [12] and FTP blood [13] are all utilized for the determination of omega-3 status. Furthermore, omega-3 blood biomarkers reported in the literature are variable and fatty acid storage stability may be dependent on the specific biomarker assessed [14]. Reported omega-3 blood biomarkers include but are not limited to the percentage of % EPA þDHA [1,15], the percentage of omega-3 highly unsaturated fatty acids (HUFA) in total HUFA [16], ratio of omega-6 polyunsaturated fatty acids (PUFA) to omega-3 PUFA (n-6/n-3 ratio) [17] and ratio of EPA to arachidonic acid (ARA, 20:4n-6) [18]. In fatty acid research, and particularly with omega-3 fatty acids and blood biomarker assessments, understanding storage stability is imperative as omega-3 fatty acids contain between three to six double bonds making them highly susceptible to degradation through lipid peroxidation pathways [19]. The purpose of this review is to provide a more complete understanding of the stability of fatty acids during long-term storage in plasma, erythrocytes and whole blood stored in both cryovials and on chromatography paper, and to discuss the mechanisms of degradation and treatments available to minimize these losses during storage. Specifically, we will examine mechanisms related to fatty acid and lipid peroxidation, strategies employed to prevent fatty acid peroxidation, the present literature on sample storage in plasma, erythrocyte and whole blood fractions, and mechanisms related to fatty acid degradation during storage. Additional focus will be given to storage of whole blood collections due to their recent increase in use and the relatively few prior studies assessing storage stability in these blood fractions. Additional mechanistic focus will be placed on the relatively recent understanding that storage of these whole blood fractions in addition to erythrocytes at  20 °C results in rapid degradation in PUFA.

2. Lipid peroxidation Whether bound or unbound to a glycerol or other lipid backbone molecules, PUFA are highly susceptible to peroxidation reactions as a result of the attack of free radicals. Lipid

H (1)

R COOH

+

.OH Initiation

(2)

H2O

.

R COOH

O2

.

O O (3)

+

(5) Propagation R COOH

H R COOH

(4)

H O O

R COOH

Fig. 1. Mechanism of free radical induced fatty acid peroxidation. (1) Unsaturated fatty acid reacts with hydroxyl radical and yields water to form (2) lipid radical which reacts with oxygen to form (3) lipid peroxyl radical that reacts with a fatty acid to form (4) lipid peroxide. The last reaction forms a new lipid radical that (5) propagates back to step (2) and re-enters the pathway.

peroxidation occurs when a highly reactive hydrogen atom is removed from the methylene group (–C ¼C–) of PUFA by a free radical resulting in the formation of a lipid peroxyl radical in its place [20]. This lipid peroxyl can react further with another PUFA to form the lipid peroxide in a continuous chain of lipid peroxidation reactions [19] (Fig. 1). However, lipid peroxidation reactions slow down when the ratio of proteins to fatty acids becomes high, in which case proteins become more susceptible to free radical attack [19]. A free radical is any molecule that contains an unpaired electron such as low activity molecules like the superoxide radical (∙O2) and highly reactive molecules such as the hydroxyl radical (∙OH) [21]. The greater the number of double bonds present in a fatty acid, the greater the reactivity of the free radical to the reactive hydrogens on the methylene groups, thus explaining why fatty acids with more double bonds degrade at a faster rate than monounsaturated (one double bond) or saturated fatty acids (no double bonds) [19]. Other mechanisms of PUFA peroxidation exist, particularly in erythrocytes that make up nearly half of a whole blood sample and are relatively high in iron (Fe). In healthy cells, approximately 3% of the hemoglobin–ferrous iron complex (Hb– Fe2 þ ) is converted to Hb–ferric iron (Fe3 þ ) by O2. This conversion results in the production of the ∙O2 radical [22–24] that can subsequently attack PUFA resulting in lipid peroxidation. Additionally, Fe2 þ can accept a proton from H2O2 that results in the formation of the more potent ∙OH radical [19] (Fig. 2). The formation of Fe3 þ on its own is able to act directly on PUFA to accept an electron from double bonds and also yields lipid radicals [25]. Lipid radicals react with O2, and the resultant lipid peroxyl radicals formed from PUFA can be converted to lipid hydroperoxides by vitamin E for removal; however, if efficient removal does not occur, then the hydroperoxides can decompose to form more free radicals in the presence of iron, thereby, further exacerbating oxidative damage

A.H. Metherel, K.D. Stark / Prostaglandins, Leukotrienes and Essential Fatty Acids 104 (2016) 33–43

35

H (1)

R COOH

+ 3+

Fe

.OH +

Fe2+ + O2

.O

Fe3+

Fe2+

Initiation

(2)

Fe2+ + H2O2

.

R COOH

2

+ Fe3+

O2

.

O O

(5) Propagation R COOH

(3)

+ H R COOH

(4)

H O O +

.

R COOH

Fe3+

O O R COOH

Fig. 2. Mechanism of iron induced lipid peroxidation. (1) Polyunsaturated fatty acid reacts with Fe3 þ and yields Fe2 þ that reacts with either O2 or H2O2 to form hydroxyl or oxygen radicals that can cause further lipid peroxidation and yield Fe3 þ that can (1) either react again with polyunsaturated fatty acids or (4) react with lipid hydroperoxides to reform lipid peroxyl radicals that (5) propagates back to step (2) and re-enters the reactions.

[26]. The iron-mediated pathway is complex with numerous oxidative species becoming recycled within the pathway (Fig. 2).

3. Strategies to prevent fatty acid losses during storage A variety of strategies for the prevention of fatty acid losses exist and can be employed throughout the analytical process. The use of butylated hydroxytoluene (BHT) is the most common of these strategies. BHT is a universal proton donor that can neutralize free radicals and prevent them from accepting protons from the methylene group of PUFA (Fig. 3) [27,28]. Since its discovery in improving PUFA yields [29], BHT has become a frequent additive in fatty acid analysis [30], and as discussed throughout this review is a popular additive to blood samples for preventing PUFA degradation during long-term storage [2,31–34]. BHT has also been shown to prevent Fe3 þ -induced PUFA peroxidation [26,35,36] and is indicative of its universal antioxidant capabilities. During storage at sub-zero temperatures [37] and even at 4 °C [38,39], erythrocytes will lyse releasing iron that, as previously discussed, can initiate fatty acid peroxidation reactions. Deferoxamine, an iron chelator, has previously been used for the prevention of fatty acid peroxidation in erythrocytes [34,40] and platelets [41,42]. When deferoxamine binds specifically to Fe3 þ (Fig. 4), the Fe3 þ is prevented from reacting with PUFA thereby preventing breakdown of PUFA and formation of lipid peroxidation products [19]. Recently, glycerol treatment of erythrocytes and whole blood has been used to reduce fatty acid peroxidation during storage at  20 °C by lowering the freezing point of samples and preventing freeze-thaw induced hemolysis [37]. The same study also demonstrated the efficacy of blood drying for the reduction of fatty acid losses through the removal of water from

the samples. In the absence of water, freeze-induced expansion, and in turn, mechanical stress causing lysis is significantly reduced. Nitrogen purging of headspace [31,34] or nitrogen bubbling of samples [43,44] are additional popular options for the prevention of fatty acid degradation during storage. Each can easily be performed just prior to storage without the addition of chemicals such as BHT or deferoxamine to the blood sample. Nitrogen displaces oxygen from the sample prior to storage thereby preventing oxygen-initiated fatty acid peroxidation reactions. BHT, iron chelation and nitrogen treatment are the three of the most common strategies to prevent fatty acid peroxidation during storage, and the benefits of each during the storage of plasma/serum, erythrocytes and whole blood will be discussed in detail in the following sections.

4. Fatty acid stability during storage 4.1. Plasma/serum In total, seven studies have examined plasma and/or serum storage stability while reporting individual changes in fatty acid compositions (Table 1). None of these studies utilized an antioxidant during storage and three of them stored their samples either under a nitrogen headspace [34] or with nitrogen bubbled into the sample [43,44]. Storage periods ranged between 24 h [44] and 10 years [45], and storage temperatures include room temperature and 4 °C [44,46],  20 °C [34,43,44,47],  60 °C [46] and  80 °C [45,48]. Interestingly, none of the studies reviewed have assessed fatty acid stability in plasma total lipid extract (TLE), although two of the studies assessed the stability of fatty acids in

36

A.H. Metherel, K.D. Stark / Prostaglandins, Leukotrienes and Essential Fatty Acids 104 (2016) 33–43

O.

CH3

OH

CH3

CH3

H3C

CH3

H 3C

CH3

CH3

CH3

H3C

CH3

H3C

CH3

.OH

.O2

CH3

O

CH3

CH3

H3C

CH3

H3C

CH3

CH 3

OOH

Fig. 3. Butylated hydroxytoluene and antioxidant products.

O O

N NH

HN

O O

N

O Fe

O

O O

N CH3

NH 2 Fig. 4. Iron chelation by deferoxamine.

multiple lipid fractions including phospholipids (PL), triacylglycerols (TAG), cholesteryl esters (CE) and NEFA [43,46] while two others assessed PL, TAG and CE but not NEFA [45,47]. Based on these studies, plasma and serum TAG and CE are stable at room temperature for at least six days but not PL and NEFA [45]. All lipid fractions are stable at 4 °C for at least six days [46], however, NEFA have been shown to increase beginning after only two days of storage [44]. The most significant changes at room temperature appear to occur from increases in NEFA linoleic acid (18:2n-6, LNA) and ARA and decreased phospholipid LNA [46]. These changes may be due to the presence of active phospholipase enzymes at room temperature. One previous study in human platelets suggests that phospholipase A2 (PLA2); the enzyme that cleaves fatty acids from the sn-2 position in phospholipids, remains active during storage at room temperature with accompanying accumulation of ARA within as little as 12 h of storage [49]. In fact, total serum NEFA increase between 24 and 48 h of storage at room temperature [50], further suggesting that NEFA may be released from phospholipid molecules when stored at room temperature. An additional study demonstrates an increase in lyso-phosphatidylcholine (lyso-PC) as a result of fatty acid

A.H. Metherel, K.D. Stark / Prostaglandins, Leukotrienes and Essential Fatty Acids 104 (2016) 33–43

Table 1 Previous studies assessing fatty acid stability in plasma/serum during storage.

Table 2 Previous studies assessing fatty acid stability in erythrocytes during storage.

Lipid Fraction

Temperature Storage PUFA Study Citations Conditions Stability Length

Lipid Fraction

CE, PL, NEFA & TAG

 20 °C

EDTA and N2

Z1 y

1y

[43]

TLE

NEFA

RT

None

6h

24 h

[44]

4 °C

None

48 h

96 h

 20 °C

N2

12 d

30 d

RT

None

o6 d

6d

4 °C

None

Z6 d

6d

 60 °C

None

Z1 y

1y

CE, PL & TAG

 20 °C

None

o3 y

3y

[47]

PL

 20 °C

N2

Z4 w

4w

[34]

TAG

 80 °C

None

Z4 y

4y

[48]

CE, PL & TAG

 80 °C

None

Z 10 y

10 y

[45]

Temperature (°C)

Storage conditions

PUFA stability

Study length

Citations

Saline

1d

6d

[52]

None

o6 m

6m

[31]

None

13 d

30 d

[53]

None

4w

17 w

[32]

BHT

Z 17 w

Saline

3d

90 d

[37]

Glycerol

90 d

None

o 60 d

60 d

[54]

Na2S2O4

Z 60 d

 70

None

Z1 y

1y

[55]

 80

BHT and N2

Z2 y

2y

[31]

 20

N2

o4 w

4w

[34]

 50

N2 þ BHT or DFO

Z1 y

1y

 80

None

Z4 y

4y

4  20

CE, PL, NEFA & TAG

37

[46]

 25

PL CE, cholesteryl ester; PL, phospholipid; NEFA, non-esterified fatty acid; TAG, triacylglycerol; RT, room temperature; EDTA, ethylenediaminetetraacetic acid; N2, nitrogen; h, hours; d, days; w, weeks; y, years. PC

hydrolysis in the sn-2 position during plasma storage at room temperature for 5 days, however, neither the presence of a soluble (sPLA2) or cytosolic (cPLA2) phospholipase inhibitor could prevent the accumulation of lyso-PC [51], suggesting an alternative mechanism for fatty acid hydrolysis. Conversely, the aforementioned study showed no effect on lyso-PC accumulation during storage of plasma at 4 °C for 7 days. In support of this finding, storage of plasma at 4 °C shows no significant changes in profiles for up to a minimum of 6 days in CE, PL, NEFA and TAG [46]. Further stability has been demonstrated in various plasma/serum lipid fractions at  20 °C for a minimum of four weeks with nitrogen [34] and a minimum of 1 year without [43], at  60 °C for one year [46] and at  80 °C for up to ten years or more [45,48]. During storage at  20 °C, fatty acids may be stable for a minimum of one year for each of the four major lipid fractions [43] and for less than three years in phospholipids, TAG and CE [47]. Strangely, one prior study determined that plasma NEFA were stable at 20 °C for only 10 days, however, the study was only 30 days in length of which no data was presented for this time point [44]. Not surprisingly, plasma and serum fatty acids are very stable when stored between  60 °C and 80 °C as none of the studies were long enough to determine significant fatty acid degradation after one year at  60 °C in all lipid fractions [46], and after four years in TAG [48] and 10 year in PL, CE and TAG [45] at 80 °C. During cold storage at temperatures between 4 °C and 80 °C plasma/serum PUFA appear stable during various storage conditions for as long as 6 days at 4 °C, 1–3 years at  20 °C, at least 1 year at  60 °C and at least 10 year at  80 °C, although further assessments on stability at 4 °C is required to determine PUFA stability beyond 7 days. Storage of plasma/serum at room temperature should be avoided for extended periods of time with PUFA losses beginning after approximately 6 h. 4.2. Erythrocytes In total, 10 studies have examined erythrocyte fatty acid stability during storage (Table 2). Three of these studies utilized an antioxidant during storage [31,32,34], two of which also stored

[48]

TLE, total lipid extract; PL, phospholipid; PC, phosphatidylcholine; BHT, butylated hydroxytoluene; DFO, deferoxamine; Na2S2O4, sodium sulfate; N2, nitrogen; h, hours; d, days; w, weeks; m, months; y, years.

erythrocytes under nitrogen [31,34]. Storage periods ranged between six days and four years, and storage temperatures include 4 °C [52],  20 °C [31,32,34,37,53],  25 °C [54],  50 °C [34],  70 °C [55] and  80 °C [31,48]. All nine of the studies assessed fatty acid compositions in either erythrocyte TLE [31,32,34,37,52,53,55], total phospholipids [34,54] or total phosphatidylcholine [48]. Fatty acid assessments in erythrocytes stored at 4 °C reveals that after only one day ARA is degraded by 24% from baseline, and DHA is degraded by 43% after two days of storage [52], and is in contrast to the stability of plasma/serum fatty acids for at least 6 days of storage at 4 °C [46]. As expected, and similar to storage of plasma, storage of erythrocytes at  80 °C for as long as two years in the presence of nitrogen and BHT [31] and four years with no antioxidant treatments [48] yields no significant change in the fatty acid composition in TLE and phosphatidylcholine, respectively. Storage at  70 °C also shows no significant changes in the fatty acid profile of erythrocyte total lipid extracts for as long as one year [55]. When storage at  80 °C is unavailable storage at  20 °C is often employed, however, previous fatty acid stability studies on the storage of erythrocytes reveals that significant degradation occurs at this temperature. Packed erythrocytes stored at  20 °C without additional treatments are shown to be stable for as little as 13 days by the ratio of HUFA:saturated fatty acids (SFA) [53] and up to four weeks as determined by DHA and total omega-3 PUFA in TLE [32]. In the latter study, both DHA ( 8%) and total omega-3 PUFA (  14%) are degraded significantly after nine weeks with all the other measured fatty acids including total omega-3 (  38%) and omega-6 PUFA (  32%) declining significantly after 17 weeks of storage. Lower PUFA compositions have also been demonstrated in total lipid extracts of erythrocytes when stored for 60 days at  25 °C [54] and for six months at  20 °C [31].

38

A.H. Metherel, K.D. Stark / Prostaglandins, Leukotrienes and Essential Fatty Acids 104 (2016) 33–43

These studies assessing fatty acid storage stability in erythrocytes provide clear evidence for cold storage conditions of at minimum  70 °C, with sample storage at  20 °C seriously increasing the risk of fatty acid losses. This can be problematic in field research studies where access to  70 °C to  80 °C storage is unavailable. Fortunately, efforts have been made to extend and prevent degradation during storage of erythrocytes at the more accessible 20 °C storage temperature. The addition of BHT to erythrocytes extends fatty acid stability at  20 °C from four weeks to at least 17 weeks [32], and another reducing agent, sodium hydrosulfate (Na2S2O4), has been utilized to extend the stability of erythrocyte PUFA to at least 60 days [53]. Interestingly, storage of erythrocytes under a nitrogen headspace does not appear to prevent fatty acid degradation when stored at  20 °C for four weeks, although fatty acid loss is prevented in the same study when iron chelation with deferoxamine is combined with storage at 50 °C [34]. However, this improvement in stability makes it difficult to ascertain whether the reduced PUFA losses are due to the colder 50 °C storage temperature or the presence of the iron chelator. The inability of nitrogen to protect against degradation suggests that the presence of oxygen during storage at  20 °C does not play a major role in degradation. Indeed, a recent study determined that HUFA losses during storage at  20 °C could be extended from as little as 3 days in a saline control to at least 90 days when treated with glycerol, a cryopreservant that prevents freeze-thaw induced hemolysis [37]. To our knowledge, this is the only study to assess the effects of cryopreservation on fatty acid stability during storage of erythrocytes. Additionally, it is concluded that erythrocytes stored at 4 °C [52] or  20 °C [37] with saline to replicate original hematocrit values are stable for very

short periods of one and three days, respectively. This is compared to the fatty acid stability of multiple weeks in packed erythrocyte samples containing much lower water content. As such, higher water content is expected to play a role in facilitating PUFA losses at  20 °C in blood samples containing erythrocytes; this phenomenon will be highlighted in detail in this review. 4.3. Whole blood in cryovials Compared to erythrocytes and plasma/serum, few studies have assessed fatty acid stability in whole blood stored in cryovials (Table 3). Prior to 2013, only one previous study assessed fatty acid stability in whole blood [44]. This study from 1978 determined that NEFA in whole blood increased after 1, 2 and 48 h when stored at 37 °C, room temperature and 4 °C, respectively. Recently, our lab has extensively assessed the long-term stability of PUFA in whole blood stored in cryovials [56]. This study examines the effects of temperature (room temperature, 4 °C,  20 °C and Table 4 Previous studies assessing fatty acid stability in whole blood stored on chromatography paper. Lipid Fraction

Temperature Storage conditions PUFA stability

TLE

RT

Z17 h

17 h

[63]

BHTþ EDTA

Z9 w

9w

[60]

Open Air

o3 d

4w

[14]

BHTþ Open Air

3w

4w

BHT

Z8 w

8w

BHT

o2 m

2m

o3 m

4m

BHT

Z3 w

3w

[2]

None

Z14 d

14 d

[62]

4 °C

None

o2 d

28 d

[61]

 20 °C

None

7d

RT

None

30 d

180 d

[56]

90 d

[37]

RT Temperature Storage conditions PUFA stability

Study length

Citations

NEFA

37 °C

1h

1h

[44]

RT

2h

24 h

4 °C

48 h

96 h

EDTA

3d

180 d

Heparin

30 d

Heparin þ BHT

180 d

EDTA

30 d

Heparin

30 d

Heparin þ BHT

180 d

EDTA

3d

Heparin

7d

Heparin þ BHT

180 d

EDTA

180 d

TLE

RT

4 °C

 20 °C

 75 °C

 20 °C

None

4 °C

[56]

4 °C

4 °C

 20 °C

 75 °C

None

7d

BHT

120 d

None

1d

BHT

90 d

None

180 d

BHT  20 °C

3d

Heparin

Dried

14 d

Heparin þ BHT

Dried þDFO

30 d

Dried þBHT

30 d

Dried þBHT þ DFO

30 d

3d

Glycerol

90 d

[33]

BHT

None

Saline

Citations

BHT

Table 3 Previous studies assessing fatty acid stability in whole blood stored in cryovials. Lipid Fraction

Study length

90 d

[37]

TLE, total lipid extract; NEFA, non-esterified fatty acid; EDTA, ethylenediaminetetraacetic acid; BHT, butylated hydroxytoluene; h, hours; d, days.

BHT, butylated hydroxytoluene; DFO, deferoxamine; EDTA, ethylenediaminetetraacetic acid TLE, total lipid extract; h, hours; d, days; w, weeks; m, months.

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75 °C) anticoagulant/antioxidants (ethylene diamineteraacetic acid (EDTA), heparin and heparin þBHT) and time (1–180 days) on the degradation of EPA þDHA (μM concentrations). To summarize, in EDTA-treated blood, EPA þDHA remained stable for only 3 days at room temperature and  20 °C and 30 days at 4 °C. Treatment with heparin extends stability to 30 days at room temperature and 7 days at  20 °C, however, no effect of heparin treatment is shown at 4 °C. Interestingly, we have shown that whole blood stored at  20 °C and treated with glycerol – a cryopreservant used to prevent sample freezing – extends PUFA stability for at least 90 days [37], a finding that will be discussed in greater detail later. Heparin has previously been shown to protect against Fe3 þ -catalyzed α-linolenic acid (18:3n-3) peroxidation [57,58] and supports the collection of blood with heparin to minimize fatty acid degradation during handling and storage procedures. EDTA also functions as an iron (Fe3 þ ) chelator [25], however, it is noted that the net effect of the Fe3 þ –EDTA complex is a balance between a reduced initiation of lipid peroxidation and an acceleration of chain propagation and branching reactions (Fig. 4). As such, iron chelation by EDTA appears to have minimal effects on PUFA losses during storage under our whole blood storage study's experimental conditions, and EDTA-chelation has led to contradictory reports on the efficacy of EDTA's effects on preventing lipid peroxidation [59]. Both EDTA and heparin-treated blood do not degrade during 180 days of storage at 75 °C, and treatment of blood with heparin þBHT prevents degradation at all storage temperature. 4.4. Whole blood on chromatography paper To date, eight studies have assessed the stability of PUFA in whole blood on chromatography paper (indicative of fingertip prick capillary blood) during storage at room temperature [14,33,56,60], 4 °C [2,33,56,61,62],  20 °C [37,56,61] and  75 °C [56] (Table 4). Of these eight studies, however, four were performed for the study validation purposes and were therefore not exhaustive [2,61–63], and another by our lab was designed to assess the mechanisms involved in rapid PUFA losses during storage at  20 °C [37]. Combined, the aforementioned four studies determine the stability of PUFA in blood on paper stored at 4 °C to be less than two days [61] and at least 14 days [62] without antioxidant treatment, and for at least 17 h at room temperature [63] and three weeks at 4 °C following [2] treatment of chromatography paper with BHT. Stability of PUFA at  20 °C was also determined to be only 7 days [61]. Additional studies analyzing blood stored on paper have determined the PUFA stability at room temperature to be less than two months in the presence of BHT [33] and at least 2 months with BHT and EDTA, and for at least 3 weeks at 4 °C in the presence of 50 μg of BHT added directly to the chromatography paper prior to blood saturation of the paper. Naturally occurring factors in the blood can play a role in enhancing PUFA degradation including iron content [64], while αtocopherol in plasma and proportion of omega-3 in erythrocytes of Icelandic women [65] and increased vitamin E levels in piglets have been found to help prevent omega-3 PUFA peroxidation [66]. Studies on blood stored on paper that focus primarily on changes in various PUFA have only recently become available in the literature. Assessment of dried blood spots (DBS) on chromatography paper treated with 50 μg of BHT reveal that each of the % of ARA, EPA and DHA in total fatty acids decrease significantly after only 48 h of storage at 4 °C [61]. Storage of the same samples at 20 °C delays the onset of degradation to 14 days of storage for both % EPA and % DHA, and % ARA does not change during 28 days of storage [61]. Storage of blood samples, and in particular, DBS on chromatography paper at room temperature can reduce the requirement of cold storage conditions during field research

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where access to cold storage conditions is either unavailable or unpractical. DBS stored at room temperature in open air or in sealed containers and with or without the presence of BHT on paper has been assessed previously [14]. Not surprisingly, when stored in open air, individual HUFA (expressed as % fatty acid in total fatty acids) are stable for less than 3 days when untreated. Stability of the individual HUFA is extended to 21 days when chromatography paper is pre-treated with BHT prior to blood saturation. Furthermore, when BHT-treated DBS are stored in sealed test tubes no measurable losses in HUFA composition are demonstrated for at least 8 weeks of storage, while no further time points were assessed. Recently, an exhaustive approach to assessing the stability of whole blood samples stored both in cryovials and on chromatography paper has been published [56]. Variables assessed for blood stored on paper include time (1–180 d), temperature (room temperature, 4 °C,  20 °C and  75 °C), antioxidant (with or without BHT) and omega-3 status (low versus high). Of note, this study did not utilize DBS for storage of blood samples, but did allow whole blood to fully saturate into the paper prior to placing them under storage. Not surprisingly, storage of these whole blood samples on chromatography paper are most stable when stored at  75 °C, demonstrating no significant decline in EPA þ DHA (μmol/L) with or without BHT in low and high omega-3 blood. Interestingly, it is determined that the decline in EPA þDHA may occur at a slower rate in blood samples with higher omega-3 content, particularly during storage at 4 °C (30 days vs. 7 days) and  20 °C (1 day vs. o1 day). Without the presence of BHT, storage stability is the shortest at  20 °C (1 day) followed by 4 °C (7 days) and room temperature (90 days). BHT pre-treatment extends stability to 90 days at  20 °C and 120 days at 4 °C with no effect at room temperature (90 days). This is contrary to the previous study at room temperature demonstrating degradation after on 48 h [61] and suggests a role for the storage container and/or blood volume used, as the former used polyethylene bags and 50 μL of blood while the latter used glass test tubes and 25 μL of blood [14,56]. The saturation of 50 μL of blood versus 25 μL is expected to yield more blood surface area that is exposed to air, and previous evidence suggests increasing surface area can increase PUFA degradation [53].

5. Mechanisms of fatty acid losses during storage Generally, plasma/serum is the most stable blood fraction as determined by PUFA changes during long-term storage. Erythrocyte PUFA are significantly less stable during storage compared with plasma. This is particularly evident during storage at  20 °C, in which PUFA content in erythrocytes are only stable for between 3 days [37] to 4 weeks [32] when pre-storage treatments such as BHT are not used. Interestingly, variable stability of PUFA during storage of erythrocytes may be due to differing aliquot volumes of erythrocytes during storage [53]. The effect of aliquot size on HUFA content during storage at  20 °C is clear, as the weekly changes in HUFA are  3.5% for 250 μL aliquots and  5.9% for 80 μL aliquots. This reduction was prevented by the inclusion of BHT in the sample during storage, and indicates the role of lipid peroxidation in the degradation of HUFA in erythrocytes stored at  20 °C. It is further hypothesized that since smaller aliquots were more susceptible to degradation, the higher surface are-to-volume ratio of these aliquots may have increased exposure to air and subsequently oxidation. In addition, it is believed that phospholipase enzymes remain active at temperatures as cold as  20 °C but not at  70 °C [67]. However, this conclusion is based on significant declines in erythrocyte phospholipid ARA levels during

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storage at  20 °C compared with  70 °C, and such a finding does not preclude other potential mechanisms of ARA loss. A second variable that appears to affect rate of PUFA degradation in erythrocytes stored at  20 °C is the water content of the stored erythrocytes. PUFA in erythrocytes stored in saline solution decline significantly in as little as 1 day at 4 °C [52] and 3 days at 20 °C [37] compared with PUFA in packed erythrocytes that decline between 13 days [53] and 4 weeks [32] at  20 °C. In light of this, increasing water content of a blood sample may negatively affect storage stability during  20 °C, and suggests that whole blood that naturally contains more water than packed erythrocytes may be a less stable blood fraction for long-term preservation of fatty acid profiles. Indeed, HUFA in whole blood collected in either EDTA or heparin-treated tubes and stored in cryovials at  20 °C degrade in as little as 3 and 7 days, respectively [56]. Taking into consideration both the stability of plasma/serum and instability of erythrocytes and to a greater extent whole blood, it is postulated that the combination of water and erythrocytes in a single fraction such as whole blood exacerbates the effect of PUFA degradation at 20 °C. Recent evidence suggests that erythrocyte rupture or hemolysis during the freezing process of blood is involved [37], and provides an explanation for the accelerated degradation of whole blood [56,61] and erythrocytes [31,32,34,53] during cold storage at 20 °C. During hemolysis, the rupturing of the erythrocyte membrane releases its contents, including hemoglobin and lipids, into the surrounding plasma and can occur both in vivo and ex vivo during handling and storage procedures [68]. Hemolysis ex vivo may be caused by factors including preparatory procedures, shear stress, bacterial contamination, membrane defects and deformability, osmotic and pH changes, blood age, leukocyte presence, drugs, irradiation, storage time and temperature [69]. Blood storage has been shown to significantly alter erythrocyte membrane integrity and flow properties resulting in an increase in the levels of free hemoglobin in plasma [38,70,71]. Interestingly, research from our lab determined that blood samples stored from an individual with a higher omega-3 status may delay or slow down the degradation process [56]. Initially, we determined that EPA þ DHA in high omega-3 blood decreased less as compared with low omega-3 blood stored on chromatography paper in both absolute (  104 μM vs.  177 μM) and relative (  14% vs.  89%) amounts from baseline to three days of storage. Fatty acid peroxidation rates increase with increasing number of double bonds, however, these results do not suggest a similar relationship with increased blood omega-3 status that is accompanied by an increase in double bonds. Peroxidation rates for various PUFA have been determined at 62 M  1 s  1 for 18:2n-6, 115 M  1 s  1 for 18:3n-3, 197 M  1 s  1 for 20:4n-6, 249 M  1 s  1 for 20:5n-3 and 334 M  1 s  1 for 22:6n-3 [72,73]. In support of these findings, osmotic fragility, a marker of hemolytic susceptibility, has been positively associated with erythrocyte n-6 PUFA content [74], and is frequently used as measure erythrocyte integrity [75,76]. In addition, humans [77] and rats [78] receiving diets high in n-3 PUFA demonstrate significantly lower osmotic fragility. These changes suggest a more stable erythrocyte membrane that is less susceptible to hemolysis as a result of fish oil supplementation. Alternatively, osmotic fragility is affected by erythrocyte vitamin E [79] levels in blood, and the generally high levels of vitamin A, D and E in fish oils [80] may also play a significant role in the prevention of HUFA degradation in blood samples following fish oil supplementation. In fact, vitamin E supplementation in pigs [81] is shown to improve oxidative stability of fatty acids in pork meat during storage. This membrane instability may provide a possible mechanism for the accelerated HUFA degradation that occurs during  20 °C storage as hemolysis can release Fe3 þ from the cell to initiate peroxidation reactions.

When the storage of blood samples for future fatty acid profiling is required, they are generally stored at  80 °C; however, when ultra-cold facilities are not available,  20 °C storage is a common alternative storage temperature. Storage temperatures below freezing generally result in significant and complete hemolysis, and although the mechanisms are poorly understood they are reviewed elsewhere [82]. Briefly, during freezing, growing extracellular ice forms channels where cells are displaced and the pressure that forms in these channels can cause extensive cell deformation and subsequently rupture or lysis. Intracellular water can also become frozen though gap junction and transmembrane protein ice crystal movement and can cause further mechanical damage to the cell. Treatment of blood samples with glycerol serves to lower the freezing point of samples thereby preventing freeze-thaw induced hemolysis [83,84], and subsequently prevents the release of free iron from hemoglobin. Furthermore, hemolysis has previously been shown to increase fatty acid peroxidation in erythrocytes [85] and the presence of antioxidants has the potential to prevent this fatty acid peroxidation from occurring [86–89]. Although hemolysis occurs during storage at both  20 °C and  75 °C, significantly altered fatty acid profiles are not shown during storage at  75 °C [37]. We hypothesize that contrary to storage at  20 °C, storage at  75 °C is sufficiently cold to completely stop all peroxidation reactions from proceeding. The effect of freeze-thaw induced hemolysis on HUFA degradation has been recently assessed by our lab during long-term storage at  20 °C [37]. Briefly, the storage of erythrocytes in the presence of the cryopreservant, glycerol, prevents significant declines in total HUFA during storage at  20 °C for at least 90 days compared to less than 3 days in saline-controlled erythrocytes. In addition, glycerol is shown to similarly protect against HUFA degradation in whole blood samples stored in cryovials. As discussed previously, the presence of glycerol in blood during cold storage lowers the freezing point of the sample to a temperature below  20 °C and prevents freeze-thaw induced hemolysis. When whole blood is stored on chromatography paper in the presence of an iron chelator, such as deferoxamine [37] or EDTA [60], this protects against HUFA decline when stored at  20 °C and room temperature, respectively, suggesting that the release of iron during hemolysis is heavily involved in the initiation of lipid peroxidation. In addition, when whole blood is dried prior to storage, the removal of water from the sample prevents freeze-thaw induced hemolysis and subsequent iron release from the cell [37]. Dried blood spotting for fatty acid profiling is a commonly used technique and its use is reported recently [90,91], although benefits of this technique are more generally attributed to improve handling of samples during transport and storage. Unfortunately, blood drying may increase HUFA degradation through other lipid peroxidation mediators as BHT is shown to provide additional protection beyond that of iron chelation alone, and therefore should not be utilized alone but in concert with chemical antioxidants [37,60]. Finally, one limitation with the present review is the challenge of comparing fatty acid determinations when different methodologies are employed. This is not unique to the examination of storage conditions, but is likely more observable due to the repeated measure of the same sample over time. As the stored samples cannot be analyzed at the same time, differences in analytical conditions can potentially influence the results. For example the stability of chemical reagents can differ over time and boron trifluoride, a common derivitizing reagent is known to have stability issues with storage [92]. It is possible that subtle differences in the practices of individual laboratories could prevent or even promote PUFA degradation, so caution is necessary when interpreting this review.

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6. Conclusions Plasma/serum is the most stable blood fraction during storage when fatty acid composition is of interest. Generally, PUFA remain at 4 °C for days and possibly longer while PUFA remain stable for up to 10 years at sub-zero temperatures. However, stability is low when stored at room temperature with degradation beginning after approximately six hours. Fatty acids in plasma/serum are however derived mainly from lipoproteins and therefore may not reflect lipid membrane bilayers in tissues [1,93]. Erythrocytes fatty acids appear to display intermediate stability with degradation beginning sooner compared to plasma. Stability has not been previously assessed at room temperature, however, PUFA degradation erythrocytes begins in days, weeks and years when stored at 4 °C, 20 °C and  80 °C, respectively. Interestingly, whole blood PUFA appear to be less stable than either plasma or erythrocyte PUFA during storage. This represents a challenge to the increasing popularity of this less technical method of both blood collection and blood analysis. FTP blood can easily be collected from a wide range of populations including more fragile individuals such as infants and the elderly, however, researchers conducting field studies must be careful to include antioxidant protection on chromatography paper if samples are to be kept at 20 °C or warmer for any significant period of time. Hemecontaining blood samples appear especially susceptible to freezethaw induced hemolysis and subsequent Fe3 þ -initiated peroxidation reactions and significant PUFA losses at  20 °C. As such, it is imperative that whole blood, FTP blood or erythrocytes are not stored at  20 °C for any period of time if fatty acid profiling is of interest. The combination of iron, water and sub-zero temperatures as low as 20 °C creates a highly favourable environment for freeze-thaw induced hemolysis and subsequent Fe3 þ -initiated fatty acid peroxidation. When  80 °C storage is not available then samples must be treated with antioxidants (i.e. BHT), iron chelators (i.e. deferoxamine), cropreservants (i.e. glycerol) or some combination of each to maintain sample integrity at storage temperatures of  20 °C or warmer.

Funding A.H.M was supported by a MITACS Elevate Postdoctoral Fellowship (IT02771) and K.D.S. was supported through a Canada Research Chair in Nutritional Lipidomics (950-228125) during the writing of this manuscript.

Authorship A.H.M. and K.D.S. participated in drafting the manuscript, revising it critically for important intellectual content and provided final approval of the version submitted.

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