Phthalate esters used as plasticizers in packed red blood cell storage bags may lead to progressive toxin exposure and the release of pro-inflammatory cytokines

Abstract

Phthalate esters (PE's) are plasticizers used to soften PVC-based medical devices. PE's are the most abundant man-made pollutants and increase the risk of developing an allergic respiratory disease or a malignancy. The leaching of PE's in donated packed red blood cells (PRBC) during storage was assessed. PRBC transfusion bags containing CPD/AS-1 (ADSOL) buffer were analyzed. Samples were collected on storage day 1 and day 42. Two PE's, di-(2-ethylhexyl) phthalate (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP), were measured by liquid chromatography coupled to mass spectrometry (LCMS). Interleukin-8 (IL-8) was measured by standard ELISA techniques. DEHP significantly increased from 34.3 microM (+/-20.0 SD) on day 1 to 433.2 microM (+/-131.2 SD) on day 42, a 12.6-fold increase. Similarly, MEHP significantly increased from 3.7 microM (+/-2.8 SD) on day 1 to 74.0 microM (+/-19.1 SD) on day 42, a 20.2-fold increase. Also, DEHP and MEHP increased the release of IL-8 from human umbilical vein endothelial cells (HUVEC). The transfusion of older units of PRBC could lead to an accumulation of PE's possibly resulting in inflammation and other effects. This accumulation could be exacerbated due to the decreased metabolism of PE's since trauma patients have a lower esterase activity, the enzymes responsible for metabolizing PE's. The effect of oxidative stress caused by PE's is discussed as a potential mechanism for increases in inflammation caused by older units of PRBC.

Keywords: blood transfusion; endothelial cells; inflammation; oxidative stress; packed red blood cells; plasticizers; trauma.

Figure 1

Chemical structure of (A) DEHP and (B) MEHP.

Figure 2

Representative mass chromatograms measuring DEHP detected in day 1 (A) and day 42 (B) supernatants collected from stored packed red blood cell (PRBC) units (N = 10). DEHP (RT = 10.5 min, [M + H⁺] = 391.28) was detected using prepared standards (1–1,000 µM). For each run, 10 µL of sample was injected onto a YMC-Pack Protein-RP HPLC column (Waters, Milford, MA) heated to 50°C. A 20 minute linear gradient from 10 to 40% solvent B using water/0.1% trifluoroacetic acid (solvent A) and acetonitrile/0.1% trifluoroacetic acid (solvent B) was utilized with a flow rate of 1 mL/min.

Figure 3

Representative mass chromatograms measuring MEHP detected in day 1 (A) and day 42 (B) supernatants collected from stored packed red blood cell (PRBC) units (N = 10). MEHP (RT = 7.4 min, [M + H⁺] = 279.16) was detected using prepared standards (1–100 µM). For each run, 10 µL of sample was injected onto a YMC-Pack Protein-RP HPLC column (Waters, Milford, MA) heated to 50°C. A 20 minute linear gradient from 10 to 40% solvent B using water/0.1% trifluoroacetic acid (solvent A) and acetonitrile/0.1% trifluoroacetic acid (solvent B) was utilized with a flow rate of 1 mL/min.

Figure 4

Total (A) DEHP and (B) MEHP in day 1 and day 42 supernatants collected from stored packed red blood cell (PRBC) units (N = 10). DEHP and MEHP levels were quantitated by liquid chromatography/mass spectrometry (LCMS) analysis. For each run, 10 µL of sample was injected onto a YMC-Pack Protein-RP HPLC column (Waters, Milford, MA) heated to 50°C. A 20 minute linear gradient from 10 to 40% solvent B using water/0.1% trifluoroacetic acid (solvent A) and acetonitrile/0.1% trifluoroacetic acid (solvent B) was utilized with a flow rate of 1 mL/min. Data are expressed in micromolar (µM) concentrations. An asterisk (*) designates significance where p < 0.05 compared to day 1 supernatants (student t-test). Significant increases in DEHP and MEHP release from the PRBC bags were observed in the day 42 samples compared with the day 1 samples.

Figure 5

Effect of DEHP and MEHP on IL-8 release in HUVEC. HUVEC were grown to near confluency (>90%) in 48-well plates in endothelial growth medium-2 (EGM-2) containing 2% fetal calf serum, hydrocortisone, human fibroblast growth factor B, vascular endothelial growth factor, recombinant insulin-like growth factor-1, ascorbate, human epithelial growth factor, gentamycin and heparin. HUVEC were treated with media only (control), DEHP, or MEHP for 24 hours in duplicate. An IL-8 ELISA was performed on the 24-hour supernatants. Data are expressed as % IL-8 release increase versus control wells. An asterisk (*) designates significance where p < 0.05 compared to control wells (student t-test). At 10, 100 and 1,000 nM MEHP, a significant increase in IL-8 release was observed. At 10 and 100 nM DEHP, a significant increase in IL-8 release was observed. This indicates the potential pro-inflammatory capability of older units of stored packed red blood cells (PRBC).