Partial characterization of lipids that develop during the routine storage of blood and prime the neutrophil NADPH oxidase

Abstract

Factors developed during the routine storage of whole blood and packed red blood cells that primed the neutrophil (PMN) reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase significantly by 2 weeks of storage, with maximal priming activity by product outdate (2.5 to 3.7 fold). These agents appeared to be generated by cellular constituents because stored, acellular plasma did not demonstrate PMN priming. The priming activity was soluble in chloroform. Priming of the oxidase by plasma and plasma extracts was inhibited by WEB 2170, a platelet-activating factor (PAF) receptor antagonist. Separation of the chloroform-soluble compounds from plasma by normal phase high-performance liquid chromatography demonstrated two peaks of priming activity at the retention times of neutral lipids and lysophosphatidylcholines (lyso-PCs) for both whole blood and packed red blood cells. Analysis of the latter peak of PMN priming by fast atom bombardment mass spectroscopy identified several specific lyso-PC species including C16 and C18 lyso-PAF. Further evaluation by gas chromatography/mass spectroscopy demonstrated that three of these species increased dramatically over product storage time, while the other two species increased modestly, and paralleled the increase in priming activity. Commercially available, purified mixtures of these lyso-PCs primed the PMN oxidase by twofold. When PMNs were incubated with this mixture of lyso-PCs, acetylated analogs of these compounds rapidly accumulated. Thus lipids, including specific lyso-PC species, develop during routine storage of cellular blood components, prime PMNs, and possibly play a role in the severe complications of transfusion therapy.

Fig. 1

Priming of the respiratory burst by plasma from stored WS (A) and PRBCs (B). The maximal rate of superoxide anion production in response to 1 µmol/L fMLP is plotted as a function of routine storage time of WB and PRBCs. Plasma isolated from WB (■) and PRBCs (●) from the weeks of storage shown was used as the priming agent of PMNs as compared with buffer controls (dashed lines). The points represent the mean of 5 units tested, with error bars representing the SEM. *Statistical significance (p < 0.05) as compared with buffer control.

Fig. 2

Priming of the respiratory burst by plasma extracts. The maximal rate of superoxide anion production by isolated neutrophils in response to 1 µmol/L fMLP is presented for each extract: 1.25% essentially fat-free albumin (vehicle) paired control (C), extracted (chloroform-methanol) plasma from both WB and PRBCs on the day of isolation (WB0, PC0), extracted plasma from WB and PRBCs on the day of product outdate (WB35, PC42). The clear bars represent the mean of the maximal rate of superoxide anion production after 5 minutes’ incubation with the chloroform-soluble extracts of 5 units of both WB and PRBCs tested on both the day of product collection and the day of outdate, plus vehicle controls, with error bars representing the SEM. The solid bars represent identical neutrophils pretreated wish 400 µmol/L WEB 2170 to block the PAF receptor before incubation with albumin controls or the chloroform-soluble extracts. *Statistical significance (p < 0.05) as compared with both the paired vehicle control and extracted plasma from both PRBCs and WB on the day of product collection.

Fig. 3

The priming activity of plasma lipids on the day of outdate from pooled units of PRBCs separated by phospholipid classes by using normal phase HPLC, The bottom line demonstrates the separation of tritiated phospholipid standards. NL, Neutral lipids; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphoinositol; PC, phosphatidylcholine; L-PC, lysophosphatidylcholine. PMN priming activity was found in the plasma from stored PRBCs on the day of outdate at (A) the retention time of neutral lipids (NL) and at (B) the retention time of lysophosphatidylcholines.

Fig. 4

The concentration of lyso-PC in plasma from PRBCs as a function of routine storage time in weeks. The species name refers to the ether-linked side chains on the sn-1 carbon. A, The increase of the C₁₆ lyso PAF concentration; B, the increase in the 1-o-palmitoyl lyso-PC concentration.

Fig. 5

The accumulation of PAF and 1-o-acyl PAF analogs from 10⁷ neutrophils incubated with purified mixtures of commercially obtained lysophosphatidylcholines and assayed at various times. The species name refers to the 1-o-alkyl or 1-o-acyl-linked side chain on the sn-1 carbon. Acetyiation of these compounds occurred on the sn-2 carbon. A, The accumulation of the 1-o-palmitoyl lyso-PC and C₁₆ lyso-PAF. B, The accumulation of 1-o-stearoyl lyso-PC.