Assay of erythrocyte membrane fatty acids. Effects of storage time at low temperature

Marino, L.; Maffettone, Ada; Cipriano, Paola; Celentano, Egidio; Galasso, Rocco; Iovine, C; Berrino, Franco; Panico, Salvatore

doi:10.1007/bf02874182

Cited by 17 publications

(9 citation statements)

References 13 publications

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“…When our samples were stored in methanol containing BHT (0.05 mg/ml of RBC) for 8 months at −80°C, most of the fatty acid proportions did not change to an appreciable extent from pre-storage values. This is comparable with other published RBC conservation methods, which stored samples for 12 months or longer [14], [15], [16], [17]. However, a non significant trend towards a selective degradation of DHA was observed, and losses might become significant after 12 months of storage.…”

Section: Discussionsupporting

confidence: 89%

“…Fatty acids of RBC samples are stable at temperatures below −50°C with or without free radical scavenging or iron binding agents [15]. Treating the washed RBC samples prior to freezing seems unnecessary, but when adding BHT a solvent is required as this antioxidant is insoluble in water.…”

Section: Discussionmentioning

confidence: 99%

“…However, a non significant trend towards a selective degradation of DHA was observed, and losses might become significant after 12 months of storage. Increasing the BHT concentration in the sample [15] or excluding the hemolysis of RBC with distilled water prior to freeze storage [14], [17] might improve the DHA stability, but this needs to be determined for GPL bound DHA.…”

Section: Discussionmentioning

confidence: 99%

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Efficient and Specific Analysis of Red Blood Cell Glycerophospholipid Fatty Acid Composition

Klem¹,

Klingler²,

Demmelmair³

et al. 2012

PLoS ONE

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BackgroundRed blood cell (RBC) n-3 fatty acid status is related to various health outcomes. Accepted biological markers for the fatty acid status determination are RBC phospholipids, phosphatidylcholine, and phosphatidyletholamine. The analysis of these lipid fractions is demanding and time consuming and total phospholipid n-3 fatty acid levels might be affected by changes of sphingomyelin contents in the RBC membrane during n-3 supplementation.AimWe developed a method for the specific analysis of RBC glycerophospholipids. The application of the new method in a DHA supplementation trial and the comparison to established markers will determine the relevance of RBC GPL as a valid fatty acid status marker in humans.MethodsMethyl esters of glycerophospholipid fatty acids are selectively generated by a two step procedure involving methanolic protein precipitation and base-catalysed methyl ester synthesis. RBC GPL solubilisation is facilitated by ultrasound treatment. Fatty acid status in RBC glycerophospholipids and other established markers were evaluated in thirteen subjects participating in a 30 days supplementation trial (510 mg DHA/d).OutcomeThe intra-assay CV for GPL fatty acids ranged from 1.0 to 10.5% and the inter-assay CV from 1.3 to 10.9%. Docosahexaenoic acid supplementation significantly increased the docosahexaenoic acid contents in all analysed lipid fractions. High correlations were observed for most of the mono- and polyunsaturated fatty acids, and for the omega-3 index (r = 0.924) between RBC phospholipids and glycerophospholipids. The analysis of RBC glycerophospholipid fatty acids yields faster, easier and less costly results equivalent to the conventional analysis of RBC total phospholipids.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Efficient and Specific Analysis of Red Blood Cell Glycerophospholipid Fatty Acid Composition

Klem¹,

Klingler²,

Demmelmair³

et al. 2012

PLoS ONE

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show abstract

“…Previously, decreases in the PUFA content of erythrocytes are prevented for up to 4 weeks when stored at −20°C [13,14], and for up to 2 years at −80°C [15] in the absence of an antioxidant. However, access to −80°C storage is often limited outside of research institutions.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2013

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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.

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“…Levels of these omega-3 PUFAs can therefore be assumed to reflect the consumption of fish as a food group (Arab, 2003). While the most commonly used biomarker medium for validation studies of fish consumption has been plasma phospholipid PUFAs, erythrocyte membrane (RBC) measures may be more appropriate because erythrocytes are incapable of de novo fatty acid synthesis or modification of PUFAs to form EPA and DHA (Di Marino et al, 2000) and are less subject to rapid fluctuations of PUFA content caused by fish meals (Arab, 2003).…”

Section: Introductionmentioning

confidence: 99%

A valid semiquantitative food frequency questionnaire to measure fish consumption

2007

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Objective: To validate a detailed semiquantitative food frequency questionnaire designed to measure habitual fish and seafood consumption. Design: Cross-sectional validation study using an independent biomarker of fish consumption. Setting: Perth metropolitan area, Western Australia. Subjects: Ninety-one healthy volunteers of both sexes aged 21-75 years. Methods: Participants completed the questionnaire and provided a fasting blood sample for erythrocyte membrane omega-3 fatty acid (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) analysis. The questionnaire was then validated by linear regression analysis of EPA and DHA levels on categories of fish and seafood and overall consumption, adjusted for age, sex, smoking status, body mass index (BMI) and alcohol intake. Results: Regression coefficients were statistically significant for most fish and seafood items with both EPA and DHA. The strongest association was observed between oily fish and EPA, whereas no significant association was observed between lean fish and omega-3 fatty acids. Variation in omega-3 fatty acids was best accounted for by a model containing variables representing different categories of fish and seafood consumption (R 2 0.484), rather than a single variable representing overall fish and seafood consumption (R 2 0.313).Conclusions: This study confirms that the varying content of omega-3 fatty acids in foods are reflected in omega-3 biomarkers, and that the questionnaire is a valid measure of fish consumption that enables differentiation between cooking and processing methods and oily versus lean fish intake.

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Assay of erythrocyte membrane fatty acids. Effects of storage time at low temperature

Cited by 17 publications

References 13 publications

Efficient and Specific Analysis of Red Blood Cell Glycerophospholipid Fatty Acid Composition

Efficient and Specific Analysis of Red Blood Cell Glycerophospholipid Fatty Acid Composition

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

A valid semiquantitative food frequency questionnaire to measure fish consumption

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