Hypochlorous acid decreases antioxidant power, inhibits plasma membrane redox system and pathways of glucose metabolism in human red blood cells

Abstract

Hypochlorous acid (HOCl) is generated at a high concentration by activated neutrophils at sites of inflammation in a myeloperoxidase catalyzed reaction. The increased and sustained production of HOCl at inflammatory sites may lead to tissue injury and this process is believed to play an important role in the progression of several diseases like chronic inflammation, atherosclerosis and some types of cancers. We have examined the effect of HOCl on human red blood cells (RBCs) under in vitro conditions. Treatment of RBC with different concentrations of HOCl (0.05-2.5 mM) at 37°C resulted in decreased activities of major antioxidant enzymes while the antioxidant power of RBC was weakened, as shown by lowered metal-reducing and free radical quenching ability of HOCl treated cells. RBC plasma membrane redox system was also inhibited suggesting membrane damage. The enzymes of glucose metabolism were inhibited indicating deranged energy metabolism. Electron microscopic images showed gross morphological changes in HOCl treated RBC. These results show that HOCl causes major alterations in the cellular antioxidant defense system and inhibition of glycolytic pathways, which increase the susceptibility of RBC to oxidative damage.

Keywords: HOCl; PMRS; RBC; SEM; antioxidant power; oxidative damage.

Graphical abstract

Figure 1

Effect of HOCl on (A) hydrogen peroxide (H₂O₂) and (B) nitrite (NO₂⁻) levels in RBC. H₂O₂ and NO₂⁻ levels were determined in hemolysates from control and HOCl treated RBC. ^*Significantly different from control.

Figure 2

Effect of HOCl on metal reducing power of RBC. (A) FRAP and potassium ferrocyanide assays (B) CUPRAC and phosphomolybdenum assays. These assays were done using hemolysates from control and HOCl-treated RBC. Except for 0.05 mM HOCl, all other results were significantly different for HOCl treated RBCs as compared to control.

Figure 3

Effect of HOCl on DPPH and ABTS radical scavenging ability of RBC. The DPPH and ABTS assays were done using hemolysates from control and HOCl-treated RBC. TE: Trolox equivalent.

Figure 4

Effect of HOCl on PMRS of RBC determined by (A) ferricyanide and (B) ABTS^* radical scavenging methods and (C) AFR reductase. ^*Significantly different from control. PRBC: packed red blood cells.

Figure 5

SEM images of control and HOCl treated RBC. (A) untreated RBC (control); RBC treated with (B) 0.25 mM (C) 1.0 mM and (D) 2.5 mM HOCl. Magnification is ×1000.