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

Abstract

The study of the stability of saturated mono-, or polyunsaturated fatty acids, both esterified and not esterified, in plasma, circulating cells, and tissues is extremely important to validate the use of biological samples stored at low temperature in "biological banks", which are used for experimental, observational, dietary, or pharmacological studies. Since red blood cells are easily accessible cells, they are used as a marker of less-accessible tissues, especially in large-scale epidemiological studies. Data from the literature suggest that the addition of an antioxidant and the freezing of red blood cells do not cause any variation in the fatty acid composition for a period of 2-6 months up to 1 year. We evaluated the fatty acid concentration in red blood cells isolated from venous blood samples of one subject, preserved with butylated hydroxytoluene and N2 and stored at -80 degrees C for up to 2 years. Erythrocytes of venous samples of six subjects stored at -20 degrees C for 6 months without butylated hydroxytoluene and in the presence of air were used for comparison purposes. Our data demonstrate that a long storage time (2 years) does not significantly influence the erythrocyte fatty acid concentration when using very low temperatures (-80 degrees C) and antioxidants (butylated hydroxytoluene) in the presence of N2.

EPA and DHA Levels in Whole Blood Decrease More Rapidly when Stored at −20 °C as Compared with Room Temperature, 4 and −75 °C

“…Freezing at -20°C is often considered an alternative if -75°C storage is not available, but our findings indicate that the storage of blood samples at -20°C is the most detrimental to HUFA stability even in comparison to refrigeration at 4°C and storage at room temperature. The degradation of n-3 HUFA at -20°C has been reported previously in erythrocyte samples [16][17][18] and in FTP samples stored in the presence of BHT for 28 days [5]. Although the focus of present study was restricted to one subject, we confirmed excessive degradation of EPA ?…”

“…In the heparin ? BHT condition, 50 lg of BHT in methanol (2 lg/lL) was added to the aliquot just prior to storage [16][17][18]. This storage condition was designed to provide maximum protection against fatty acid peroxidation in which peroxidation protection is provided by both BHT and heparin [19,20].…”

Effects of storage time and added antioxidant on fatty acid composition of red blood cells at −20°C

“…On the other hand, addition of BHT did preserve FA proportions and concentrations of venous RBC for at least 17 wk at -20°C. BHT has been shown to preserve FA composition of RBC for at least 1 yr at lower temperature (7,8).…”

“…Storage of RBC samples for more than 4 wk before FA analysis is often unavoidable, but lower proportion of long-chain PUFA has been found after 6 mon compared to baseline (8). The stability of PUFA in RBC may be affected by the physiological or pathological state of the individual.…”

“…Some centers for health service have access to only a -20°C or -30°C freezer, and human studies have shown that venous RBC stored at -20°C without addition of antioxidant had unchanged FA composition after 4 wk compared to baseline (7). Storage of RBC samples for more than 4 wk before FA analysis is often unavoidable, but lower proportion of long-chain PUFA has been found after 6 mon compared to baseline (8). The stability of PUFA in RBC may be affected by the physiological or pathological state of the individual.…”