Oxidative Stress and Intravascular Hemolysis in Paroxysmal Nocturnal Hemoglobinuria
Authors: Osato Makiko , Nishimura Jun-ichi , Motoki Yukari , Hayashi Satoru , Ueda Yasutaka , Nojima Junzo , Kanakura Yuzuru 
Published: October 2019

Abstract
Background
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder, characterized by the complement-mediated intravascular hemolysis. Eculizumab is a humanized monoclonal antibody that binds to the human C5 protein, thereby inhibiting terminal complement-mediated hemolysis. PNH patients treated with eculizumab responded with a significant reduction in hemolysis and improved in quality of life (QOL). Recent report shows that PNH-type cells were under the oxidative stress due to elevated levels of intracellular reactive oxygen species (ROS) and/or decreased antioxidant status. Fermented papaya preparation (FPP), a dietary nutriceutical derived from carica papaya, has been shown to modulate oxidative stress damage parameters in vitro and in vivo. To clarify the pathogenetic link between oxidative stress and hemolysis in PNH, the effects of each inhibitor, eculizumab and FPP, in Japanese patients with PNH were investigated independently.
Methods
Peripheral blood and sera were obtained from patients with PNH and healthy individuals after the obtaining informed consent. The study protocol was approved by the IRB at Osaka University Graduate School of Medicine. For ROS assay, RBCs were measured using 2’-7’-dichlorofluorescin-diacetate. The oxidative stress index (OSI) was derived from the percent ratio of the Reactive Oxygen Metabolites-derived compounds test (Diacron) and the Biological Antioxidant Potential test (Diacron) using a JCA-BM1650 analyzer (JEOL). LDH, biomarker of hemolysis, was measured by routine tests. Eculizumab was administrated according to standard schedule. FPP was taken orally 18g/day for 3 month. Health-related fatigue and QOL were also assessed using the FACIT-fatigue (Version 4A) and EORTC QLQ-C30 (Version 3) instruments.
Results
PNH patients without eculizumab treatment (pts w/o ecu) showed significantly higher levels in ROS compered to healthy individuals ([mean±SD] 1.13±0.18 vs. 0.84±0.10, p=0.004, n=8). Among these patients, CD59-negative RBCs showed a significant increase in ROS compared to GPI-positive RBCs in corresponding patients (1.23±0.26 vs. 1.13±0.27, p=0.03, n=6). Similarly, the OSI level was significantly higher in pts w/o ecu than healthy individuals (1.32±0.37 vs. 0.97±0.10, p=0.022, n=9) and oxidation activity in sera was significantly higher in pts w/o ecu than healthy volunteers (361.6±91.5 vs. 285.0±31.8, p=0.039, n=9), while antioxidant activity in sera was not significantly different between these 2 groups ([2440.6±191.5 vs. 2601.5±186.6, p=0.09, n=9). Interestingly, there was no significant difference in OSI level between PNH patients on eculizumab (n=8) and healthy individuals (0.84±0.10 vs. 1.07±0.45, p=0.55). Two patients who newly started eculizumab treatment showed improvement in LDH along with ROS generation ([LDH(IU/l)] Pt.I:6082-906, Pt.II:618-211; [ROS] Pt.I:1.57-1.06, Pt.II:1.50-1.40). FPP treatment (n=2) showed little effect on LDH, while it decreased ROS (Pt.I:1.72-1.47, Pt.II:1.16-1.04) and improved the scores in FACIT-fatigue and EORTC QLQ-C30. In addition, those 2 patients showed increase in ROS after discontinuation of FPP (Pt.I:1.47-2.04, Pt.II:1.04-1.19).
Conclusion
The RBCs and sera derived from Japanese patients with PNH were highly susceptible to oxidative stress compared to healthy individuals. Eculizumab was effective in controlling the oxidative stress, in addition to the hemolysis, suggesting that elevated oxidative stress in PNH was mainly due to the complement-mediated hemolysis. Since FPP showed little effect on hemolysis but had a potential to ameliorate the oxidative stress and improved QOL, FPP could alleviate symptoms associated with oxidative stress and may contribute to the therapeutic option for supportive therapy in PNH.

Disclosures
Osato: Alexion Pharma: Research Funding. Nishimura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Hayashi:Alexion Pharma: Research Funding. Ueda:Alexion Pharma: Research Funding. Kanakura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.