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Montelukast and zafirlukast, cysteinyl leukotriene receptor antagonists (LTRAs), trigger apoptosis and inhibit cell proliferation of triple‑negative breast cancer MDA‑MB‑231 cells. By contrast, only zafirlukast induces G 0 /G 1 cell cycle arrest. The present study compared the effects of these drugs on proteins regulating cell proliferation, apoptosis, autophagy, and endoplasmic reticulum (ER) and oxidative stress using reverse transcription‑quantitative PCR, western blotting and flow cytometry. The expression of proliferating markers, Ki‑67 and proliferating cell nuclear antigen, was decreased by both drugs. Zafirlukast, but not montelukast, decreased the expression of cyclin D1 and CDK4, disrupting progression from G 1 to S phase. Zafirlukast also increased the expression of p27, a cell cycle inhibitor. Both drugs decreased the expression of anti‑apoptotic protein Bcl‑2 and ERK1/2 phosphorylation, and increased levels of the autophagy marker LC3‑II and DNA damage markers, including cleaved PARP‑1, phosphorylated (p)‑ATM and p‑histone H2AX. The number of caspase 3/7‑positive cells was greater in montelukast‑treated cells compared with zafirlukast‑treated cells. Montelukast induced higher levels of the ER stress marker CHOP compared with zafirlukast. Montelukast activated PERK, activating transcription factor 6 (ATF6) and inositol‑requiring enzyme type 1 (IRE1) pathways, while zafirlukast only stimulated ATF6 and IRE1 pathways. GSK2606414, a PERK inhibitor, decreased apoptosis mediated by montelukast, but did not affect zafirlukast‑induced cell death. The knockdown of CHOP by small interfering RNA reduced apoptosis triggered by montelukast and zafirlukast. In conclusion, the effects on cell cycle regulator proteins may contribute to cell cycle arrest caused by zafirlukast. The greater apoptotic effects of montelukast may be caused by the higher levels of activated caspase enzymes and the activation of three pathways of ER stress: PERK, ATF6, and IRE1.

Hydroxychavicol, a primary active phenolic compound of betel leaves, previously inhibited bone loss in vivo by stimulating osteogenesis. However, the effect of hydroxychavicol on bone remodeling induced by osteoclasts is unknown. In this study, the anti-osteoclastogenic effects of hydroxychavicol and its mechanism were investigated in receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclasts. Hydroxychavicol reduced the number of tartrate resistance acid phosphatase (TRAP)-positive multinucleated, F-actin ring formation and bone-resorbing activity of osteoclasts differentiated from RAW264.7 cells in a concentration-dependent manner. Furthermore, hydroxychavicol decreased the expression of osteoclast-specific genes, including cathepsin K, MMP-9, and dendritic cell-specific transmembrane protein (DC-STAMP). For mechanistic studies, hydroxychavicol suppressed RANKL-induced expression of major transcription factors, including the nuclear factor of activated T-cells 1 (NFATc1), c-Fos, and c-Jun. At the early stage of osteoclast differentiation, hydroxychavicol blocked the phosphorylation of NF-κB subunits (p65 and Iκβα). This blockade led to the decrease of nuclear translocation of p65 induced by RANKL. In addition, the anti-osteoclastogenic effect of hydroxychavicol was confirmed by the inhibition of TRAP-positive multinucleated differentiation from human peripheral mononuclear cells (PBMCs). In conclusion, hydroxychavicol inhibits osteoclastogenesis by abrogating RANKL-induced NFATc1 expression by suppressing the NF-κB signaling pathway in vitro .

Background: The increased procoagulant platelets and platelet activation are associated with thrombosis in COVID-19. In this study, we investigated platelet activation in COVID-19 patients and their association with other disease markers., Methods: COVID-19 patients were classified into three severity groups: no pneumonia, mild-to-moderate pneumonia, and severe pneumonia. The expression of P-selectin and activated glycoprotein (aGP) IIb/IIIa on the platelet surface and platelet-leukocyte aggregates were measured prospectively on admission days 1, 7, and 10 by flow cytometry., Results: P-selectin expression, platelet-neutrophil, platelet-lymphocyte, and platelet-monocyte aggregates were higher in COVID-19 patients than in uninfected control individuals. In contrast, aGPIIb/IIIa expression was not different between patients and controls. Severe pneumonia patients had lower platelet-monocyte aggregates than patients without pneumonia and patients with mild-to-moderate pneumonia. Platelet-neutrophil and platelet-lymphocyte aggregates were not different among groups. There was no change in platelet-leukocyte aggregates and P-selectin expression on days 1, 7, and 10. aGPIIb/IIIa expression was not different among patient groups. Still, adenosine diphosphate (ADP)-induced aGPIIb/IIIa expression was lower in severe pneumonia than in patients without and with mild-to-moderate pneumonia. Platelet-monocyte aggregates exhibited a weak positive correlation with lymphocyte count and weak negative correlations with interleukin-6, D-dimer, lactate dehydrogenase, and nitrite., Conclusion: COVID-19 patients have higher platelet-leukocyte aggregates and P-selectin expression than controls, indicating increased platelet activation. Compared within patient groups, platelet-monocyte aggregates were lower in severe pneumonia patients., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Srihirun et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Capsaicin and dihydrocapsaicin (DHC) are major pungent capsaicinoids produced in chili peppers. Capsaicin has been previously shown to promote vascular health by increasing nitric oxide (NO) production and reducing inflammatory responses. While capsaicin has been extensively studied, whether DHC exerts cardiovascular benefits through similar mechanisms remains unclear. The current study aimed to investigate the direct effects of DHC on endothelial inflammation, NO release, and free radical scavenging properties. DHC at concentrations up to 50 µM did not affect cell viability, while concentrations of 100 and 500 µM of DHC led to endothelial cytotoxicity. Capsaicin decreased cell viability at concentration of 500 µM. To investigate the effects of capsaicinoids on endothelial activation, we first demonstrated that TNFα induced Ser536 phosphorylation of p65 NFκB, expressions of adhesion molecules, vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1, and IL-6 production in primary human endothelial cells. These effects were robustly abrogated by DHC. Consistently, DHC treatment led to a marked reduction in TNFα-mediated monocyte adhesion to endothelial cells. Additionally, NO production was significantly induced by DHC and capsaicin compared to vehicle control. Similar to capsaicin and vitamin C, DHC scavenged DPPH (1,1-diphenyl-2-picrylhydrazyl) free radicals in vitro. Our present study highlights the benefits of DHC and capsaicin treatment on human endothelial cells and provides evidence to support cardiovascular benefits from capsicum consumption., Competing Interests: Conflict of interest statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Pulmonary hypertension is a significant complication in thalassemia patients. Recent studies showed that inhaled nebulized nitrite could rapidly decrease pulmonary artery pressure. We conducted a multicenter, randomized, double-blind, placebo-controlled trial in thalassemia patients with symptomatic pulmonary hypertension diagnosed by right heart catheterization. Eleven patients were recruited; five were assigned to the nitrite group and six to the placebo group. Patients were treated with the optimal doses of sildenafil for pulmonary hypertension and randomly assigned into the placebo or nitrite groups. Patients in the nitrite group were given inhaled nebulized 30 mg sodium nitrite twice a day for 12 weeks. The clinical outcomes measured at week 12 were the changes in 6-min walk distance (6MWD), mean pulmonary artery pressure (MPAP), and N-terminal pro B-type natriuretic peptide. The MPAP estimated by echocardiography was significantly reduced from 33.6 ± 7.5 mmHg to 25.8 ± 6.0 mmHg (mean difference = 7.76 ± 3.69 mmHg, p = 0.009 by paired t-test). Furthermore, 6MWD was slightly increased from 382.0 ± 54.0 m to 432 ± 53.9 m (mean difference = 50.0 ± 42.8 m, p = 0.059 by paired t-test) in the nitrite group. At week 12, the nitrite group had lower MPAP than the placebo group (25.8 ± 6.0 vs. 45.7 ± 18.5 mmHg, p = 0.048 by unpaired t-test). No significant difference in 6MWD and N-terminal pro B-type natriuretic peptide between the two groups was observed at week 12. There was no hypotension or other significant adverse effects in the nitrite group., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Nitrite anion (NO 2 - ) is a circulating nitric oxide (NO) metabolite considered an endothelial function marker. Nitrite can be produced from nitrate (NO 3 - ) secreted from plasma into saliva. The nitrate reductase of oral bacteria converts salivary nitrate to nitrite, which is swallowed and absorbed into circulation. In this study, we aimed to examine the relevance between these species' salivary and blood levels. We collected three whole saliva samples (unstimulated, paraffin-stimulated, and post-chlorhexidine mouthwash stimulated saliva) and blood from 75 healthy volunteers. We measured the nitrite and nitrate by the chemiluminescence method. The nitrite levels in stimulated saliva and post-mouthwash stimulated saliva exhibited weak correlations with blood nitrite. There was no correlation between nitrite in unstimulated saliva with blood nitrite. The baseline platelet activity, determined as P-selectin expression, negatively correlated with nitrite in plasma and post-mouthwash stimulated saliva. The salivary nitrate in all saliva samples showed correlations with its plasma levels. We conclude that nitrite in stimulated saliva correlates with blood nitrite., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Colorectal cancer (CRC) is an aggressive malignancy. Critical mechanisms that support CRC progression include cell migration, invasion, metastasis, and angiogenesis, which is associated with L1 cell adhesion molecule (L1CAM) and nuclear factor-kappa B (NF-κB) signaling pathways. In this study, viability of HT-29 cells and human umbilical vein endothelial cells (HUVECs) was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, and cell apoptosis was investigated by flow cytometry assays. HT-29 cell migration and invasion were observed by wound healing and Transwell invasion assays, respectively, and tube formation of HUVECs was observed by tubulogenesis assays. L1CAM and NF-κB protein expressions in HT-29 cells treated with onion peel extract were determined by indirect immunofluorescence. Results showed that high dose treatments of onion peel extract inhibited cell viability of both HT-29 cells and HUVECs, induced HT-29 cell apoptosis, and inhibited HT-29 cell migration and invasion. Moreover, onion peel extract decreased total HUVEC tube length and, at a concentration of 10 μg/mL, showed potential to downregulate L1CAM and NF-κB. In conclusion, onion peel extract inhibits HT-29 cell growth, migration, and invasion through suppressing pathways related to angiogenesis downstream of L1CAM-activated NF-κB., Competing Interests: AUTHOR DISCLOSURE STATEMENT The authors declare no conflict of interest., (Copyright © 2021 by The Korean Society of Food Science and Nutrition. All rights Reserved.)

Background: Nitrite is a physiologic nitric oxide (NO) derivative that can be bioactivated to NO. NO has been shown to attenuate airway inflammation and enhance the anti-inflammatory effect of corticosteroids in the animal model of asthma. Here, we aimed to investigate the efficacy and safety of inhaled sodium nitrite as add-on therapy with inhaled corticosteroid (ICS) in adult patients with persistent asthma., Methods: In protocol 1, 10 asthmatic patients were administered a single dose of nebulized 15-mg sodium nitrite to assess safety, effect on lung function, and pharmacokinetics of nitrite within 120 min. In protocol 2, 20 patients were randomly assigned to a nitrite (15 mg twice daily) group or a placebo group to assess the efficacy over 12 weeks. The primary outcome was the forced expiratory volume in 1 s (FEV 1 ). The secondary outcomes were other lung function parameters, unplanned asthma-related visits at the emergency department (ED) or outpatient department (OPD), admission days, asthma control test (ACT), and safety., Results: Nebulized sodium nitrite had neither acute adverse effect nor effect on lung function test within 120 min. No blood pressure change was seen. At week 12, FEV 1 increased in the nitrite group, whereas there was no change in the placebo group. There were 5 events of asthma exacerbation, 4 ED visits, and one unplanned OPD visit in the placebo group, but none of these was noted in the nitrite group. There was no change in ACT scores in both groups. No adverse event was reported during 12 weeks in the nitrite group. There was no change in methemoglobin levels and sputum inflammatory markers., Conclusion: From our pilot trial, nebulized sodium nitrite is safe in asthmatic patients, and shows the potential to reduce asthma exacerbation compared with placebo., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Nickel, a heavy metal found in electronic wastes and fume from electronic cigarettes, induces neuronal cell death and is associated with neurocognitive impairment. Astrocytes are the first line of defense against nickel after entering the brain; however, the effects of nickel on astrocytes remain unknown. Herein, we investigated the effect of nickel exposure on cell survival and proliferation and the underlying mechanisms in U-87 MG human astrocytoma cells and primary human astrocytes. Intracellular nickel levels were elevated in U-87 MG cells in a dose- and time-dependent manner after exposure to nickel chloride. The median toxic concentrations of nickel in astrocytoma cells and primary human astrocytes were 600.60 and >1000 µM at 48 h post-exposure, respectively. Nickel exposure triggered apoptosis in concomitant with the decreased expression of anti-apoptotic B-cell lymphoma protein (Bcl-2) and increased caspase-3/7 activity. Nickel induced reactive oxygen species formation. Additionally, nickel suppressed astrocyte proliferation in a dose- and time-dependent manner by delaying G2 to M phase transition through the upregulation of cyclin B1 and p27 protein expression. These results indicate that nickel-induced cytotoxicity of astrocytes is mediated by the activation of apoptotic pathway and disruption of cell cycle regulation., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A mass of evidence has identified a promoting of nitric oxide (NO) production in endothelial cells using natural products as a potential strategy to prevent and treat hypertension. This study investigated whether the aqueous extract of Moringa oleifera leaves (MOE) could lower mean arterial pressure (MAP) and relax mesenteric arterial beds in rats via stimulating endothelium-derived NO production. Intravenous administration of MOE (1-30 mg/kg) caused a dose-dependent reduction in MAP in anesthetized rats. In rats pretreated with the NO-synthase inhibitor, N ω -nitro-L-arginine methyl ester (L-NAME, 30 mg/kg, i.v.), the effect of MOE on MAP was significantly reduced. MOE (0.001-3 mg) induced relaxation in methoxamine (10 μM) pre-contracted mesenteric arterial beds, which was abolished by endothelium denudation. This endothelium-dependent vasorelaxation was reduced by L-NAME (100 μM) or the NO-sensitive guanylyl cyclase inhibitor, 1H- [1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (10 μM). In primary human pulmonary artery endothelial cells, MOE (3-30 μg/mL) induced NO production, which was inhibited by L-NAME (100 μM) pretreatment. These findings show that MOE stimulates the endothelium-derived NO release for driving its vasorelaxation to lower arterial blood pressure. These suggest the development of MOE as a natural antihypertensive supplement., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Introduction: An increase in platelet activity is a contributing factor to vascular complications in hemoglobin E/β-thalassemia (HbE/β-thal). Plasma-free hemoglobin (Hb) increases in HbE/β-thal patients and correlates with platelet activation, but the levels of Hb-bound platelets have never been reported. In this study, we aimed to investigate the levels of Hb-bound platelets and its association with platelet activity in HbE/β-thal patients., Methods: Hb-bound platelets were measured by flow cytometry in 22 healthy subjects and 26 HbE/β-thal patients (16 nonsplenectomized and 10 splenectomized HbE/β-thal patients). Plasma Hb was measured by the chemiluminescence method based on the consumption of nitric oxide (NO) by Hb. Expression of P-selectin and activated glycoprotein (aGP) IIb/IIIa on platelets was measured by flow cytometry as a marker of platelet activity., Results: Both nonsplenectomized and splenectomized HbE/β-thal patients had higher levels of Hb-bound platelets and plasma Hb than healthy subjects. In vitro incubation of dialyzed Hb from patients with platelets of healthy subjects caused an increase in Hb-bound platelets, which was partially inhibited by anti-GPIbα antibody. Plasma Hb positively correlated with Hb-bound platelets. Platelet P-selectin expression at baseline and in response to adenosine diphosphate (ADP, 1 µM) stimulation was higher in nonsplenectomized and splenectomized HbE/β-thal patients than healthy subjects. The ADP-induced aGPIIb/IIIa expression on platelets was also higher in HbE/β-thal patients than healthy subjects. Hb-bound platelets correlated with baseline P-selectin expression and ADP-induced P-selectin expression., Conclusion: HbE/β-thal patients have increased Hb-bound platelets, which is associated with increased baseline platelet activation and reactivity., (© 2020 John Wiley & Sons Ltd.)

Inhaled sodium nitrite has been reported to decrease pulmonary artery pressure in hemoglobin E/β-thalassemia (HbE/β-thal) patients with pulmonary hypertension. This study investigated the pharmacokinetics and pharmacodynamics of inhaled nebulized sodium nitrite in 10 healthy subjects and 8 HbE/β-thal patients with high estimated pulmonary artery pressure. Nitrite pharmacokinetics, fraction exhaled nitric oxide (FE NO ), estimated right ventricular systolic pressure (eRVSP) measured by echocardiography, and platelet activation were determined. Nebulized sodium nitrite at doses used in this study (37.5 and 75 mg for healthy subjects and 15 mg for HbE/β-thal patients) was well tolerated and did not cause changes in methemoglobin levels and systemic blood pressure. Absorption of inhaled nitrite was rapid with the absolute bioavailability of 18%. In whole blood, nitrite exhibited the dose-independent pharmacokinetics with clearance (CL) of 1.5 l/h/kg, volume of distribution (V d ) of 1.3 l/kg and half-life (t 1/2 ) of 0.6 h. CL and V d of nitrite was higher in red blood cells (RBC) than whole blood and plasma. HbE/β-thal patients had lower nitrite CL and longer t 1/2 in RBC than healthy subjects. FE NO increased immediately after inhalation. Following nitrite inhalation, eRVSP remained unchanged but platelet activation was suppressed as evidenced by inhibition of adenosine diphosphate (ADP)-induced P-selectin expression and increase in phosphorylated vasodilator-stimulated phosphoprotein (P-VASP Ser239 ) in platelets. There were no changes in markers of oxidative and nitrosative stress after inhalation. Our results support further development of inhaled nebulized sodium nitrite for treatment of pulmonary hypertension in β-thalassemia., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Nitric oxide (NO) can be generated from nitrite by reductase activity of deoxygenated hemoglobin (deoxyHb) apparently to facilitate tissue perfusion under hypoxic condition. Although hemoglobin E (HbE) solutions have been shown to exhibit decreased rate of nitrite reduction to NO, this observation has never been reported in erythrocytes from subjects with hemoglobin E/ß-thalassemia (HbE/ß-thal). In this study, we investigated the nitrite reductase activity of deoxyHb dialysates from 58 non-splenectomized and 23 splenectomized HbE/ß-thal subjects compared to 47 age- and sex-matched normal subjects, and examined its correlation with platelet activity. Iron-nitrosyl-hemoglobin (HbNO) was measured by tri-iodide reductive chemiluminescence as a marker of NO generation. HbNO produced from the reaction of nitrite with deoxyHb dialysate from both non-splenectomized and splenectomized HbE/ß-thal subjects was lower than that of normal (AA) hemoglobin subjects. P-selectin expression, a marker of platelet activation, at baseline and in reactivity to stimulation by adenosine diphosphate (ADP), were higher in HbE/ß-thal subjects than normal subjects. HbNO formation from the reactions of nitrite and deoxyHb inversely correlated with baseline platelet P-selectin expression, HbE levels, and tricuspid regurgitant velocity (TRV). Nitrite plus deoxygenated erythrocytes from HbE/ß-thal subjects had a lower ability to inhibit ADP-induced P-selectin expression on platelets than erythrocytes from normal subjects. We conclude that deoxyHb in erythrocytes from HbE/ß-thal subjects has a decreased ability to reduce nitrite to NO, which is correlated with increased platelet activity in these individuals., Competing Interests: The authors have declared that no competing interests exist.

Iron chelation can improve endothelial function. However, effect on endothelial function of deferiprone has not been reported. We hypothesized deferiprone could promote nitric oxide (NO) production in endothelial cells. We studied effects of deferiprone on blood nitrite and blood pressure after single oral dose (25 mg/kg) in healthy subjects and hemoglobin E/β-thalassemia patients. Further, effects of deferiprone on NO production and endothelial NO synthase (eNOS) phosphorylation in primary human pulmonary artery endothelial cells (HPAEC) were investigated in vitro. Blood nitrite levels were higher in patients with deferiprone therapy than those without deferiprone (P = 0.023, n = 16 each). Deferiprone increased nitrite in plasma and whole blood of healthy subjects (P = 0.002 and 0.044) and thalassemia patients (P = 0.003 and 0.046) at time 180 min (n = 20 each). Asymptomatic reduction in diastolic blood pressure (P = 0.005) and increase in heart rate (P = 0.009) were observed in healthy subjects, but not in thalassemia patients. In HPAEC, deferiprone increased cellular nitrite and phospho-eNOS (Ser1177) (P = 0.012 and 0.035, n = 6) without alteration in total eNOS protein and mRNA. We conclude that deferiprone can induce NO production by enhancing eNOS phosphorylation in endothelial cells.

Pulmonary hypertension is a life-threatening complication in β-thalassemia. Inhaled sodium nitrite has vasodilatory effect on pulmonary vasculature. However, its effect on pulmonary artery pressure (PAP) in β-thalassemia subjects with pulmonary hypertension has never been reported. In this study, we investigated the change in PAP during inhalation of sodium nitrite in 5 β-thalassemia patients. We demonstrated that sodium nitrite administered by nebulization rapidly decreased PAP as measured by echocardiography and right heart catheterization. The effect of nitrite was short as PAP returned to baseline at end of inhalation. Our findings support acute pulmonary vasodilation effect of nitrite in β-thalassemia with pulmonary hypertension., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Despite a discovery of hormonal pathways regulating breast cancer, a definitive cure for the disease requires further identification of alternative targets that provide a hormone-independent support. Apart from their role in inflammatory diseases, cysteinyl leukotriene (CysLT) receptor antagonists (LTRAs) decrease the risk of lung cancer in asthma patients and inhibit tumor progression in several malignancies. In the present study, we evaluate the effects of two chemically different, clinically relevant LTRAs (montelukast and zafirlukast) in a triple negative breast cancer cell line, MDAMB- 231. We found that these two LTRAs reduced breast cancer cell viability in a dose-dependent manner with the 50% inhibitory concentration (IC50) between 5-10 μM. Although both LTRAs have several pharmacological properties in common, we noticed that montelukast mainly induced apoptosis, while zafirlukast mainly exerted its action on cell cycle. However, the precise mechanisms responsible for such different effects remain unclear. In summary, our results suggest that CysLT plays a role in proliferation and survivability of breast cancer cells in the absence of hormonal stimuli., (Creative Commons Attribution License)

Nitrite is recognized as a bioactive nitric oxide (NO) metabolite. We have shown that nitrite inhibits platelet activation and increases platelet cGMP levels in the presence of partially deoxygenated erythrocytes. In this study, we investigated the effect of nitrite on phosphorylation of vasodilator-stimulated phosphoprotein on residue serine 239 (P-VASPSer239), a marker of protein kinase G (PKG) activation, in human platelets. In platelet-rich plasma (PRP), nitrite itself had no effect on levels of P-VASPSer239 while DEANONOate increased P-VASPSer239. Deoxygenation of PRP + erythrocytes (20% hematocrit) raised baseline P-VASPSer239 in platelets. At 20% hematocrit, nitrite (10 μM) increased P-VASPSer239 in platelets about 31% at 10-20 minutes of incubation while the levels of P-VASPSer157, a marker of protein kinase A (PKA) activation, were not changed. Nitrite increased P-VASPSer239 in platelets in the presence of deoxygenated erythrocytes at 20-40% hematocrit, but the effects were slightly greater at 20% hematocrit. In conclusion, our data confirm that nitrite increases P-VASPSer239 in platelets in the presence of deoxygenated erythrocytes. They also further support the idea that partially deoxygenated erythrocytes may modulate platelet activity, at least in part, via the NO/sGC/PKG pathway from NO formed by reduction of circulating nitrite ions.

In the presence of red blood cells (RBCs), nitrite inhibits platelets through its conversion to nitric oxide (NO) by the reductase activity of partially deoxygenated hemoglobin. Inhaled sodium nitrite is being investigated as a therapy for pulmonary hypertension. Here, we measured platelet aggregation, P-selectin expression, platelet-leukocyte aggregates and phosphorylated vasodilator-stimulated phosphoprotein (P-VASP Ser239 ) following sodium nitrite inhalation in healthy subjects. In vitro incubation of nitrite with deoxygenated whole blood showed an increase in P-VASP Ser239 , which was inhibited by ODQ, a soluble guanylyl cyclase (sGC) inhibitor. Immediately and 60 min after nitrite inhalation, P-VASP Ser239 increased in platelets. Platelet aggregation, P-selectin expression, platelet-monocyte and platelet-lymphocyte aggregates decreased after inhalation. In conclusion, sodium nitrite administered to healthy subjects by inhalation can inhibit platelet activation and increase P-VASP Ser239 in platelets. Platelet inhibition by nitrite administration may be useful in disorders associated with platelet hyperactivity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Significance: Oxidative stress and generation of free radicals are fundamental in initiating pathophysiological mechanisms leading to an inflammatory cascade resulting in high rates of morbidity and death from many inherited point mutation-derived hemoglobinopathies. Hemoglobin (Hb)E is the most common point mutation worldwide. The β E -globin gene is found in greatest frequency in Southeast Asia, including Thailand, Malaysia, Indonesia, Vietnam, Cambodia, and Laos. With the wave of worldwide migration, it is entering the gene pool of diverse populations with greater consequences than expected., Critical Issues: While HbE by itself presents as a mild anemia and a single gene for β-thalassemia is not serious, it remains unexplained why HbE/β-thalassemia (HbE/β-thal) is a grave disease with high morbidity and mortality. Patients often exhibit defective physical development, severe chronic anemia, and often die of cardiovascular disease and severe infections. Recent Advances: This article presents an overview of HbE/β-thal disease with an emphasis on new findings pointing to pathophysiological mechanisms derived from and initiated by the dysfunctional property of HbE as a reduced nitrite reductase concomitant with excess α-chains exacerbating unstable HbE, leading to a combination of nitric oxide imbalance, oxidative stress, and proinflammatory events., Future Directions: Additionally, we present new therapeutic strategies that are based on the emerging molecular-level understanding of the pathophysiology of this and other hemoglobinopathies. These strategies are designed to short-circuit the inflammatory cascade leading to devastating chronic morbidity and fatal consequences. Antioxid. Redox Signal. 26, 794-813.

Chronic exposure to cadmium has been linked to brain cancers, learning disabilities and memory deficits. Previous studies of cadmium toxicity in the central nervous system report cadmium induces oxidative stress in neurons and astrocytes. In the peripheral system, cadmium promotes interleukin-6 (IL-6) and IL-8 production and release. Elevation of IL-6 expression is linked to the pathogenesis of neurodegenerative diseases and astrogliosis. IL-8 plays a role in angiogenesis of gliomas and neurodegenerative diseases. Herein, the effects of non-toxic concentrations of cadmium on the production of IL-6 and IL-8 and the underlying mechanisms were investigated. U-87 MG human astrocytoma cells and primary human astrocytes were exposed to cadmium chloride. At 24h post-exposure to 1 and 10μM, levels of intracellular cadmium in U-87 MG cells were 11.89±3.59 and 53.08±7.59μg/g wet weight, respectively. These concentrations had minimal effects on cell morphology and viability. IL-6 and IL-8 mRNA levels and secretion increased in dose- and time-dependent manners post cadmium exposure. Acute exposure to cadmium increased phosphorylation of ERK1/2, p38 MAPK, and p65 NF-κB. Pretreatment with U0126-an inhibitor of MEK1 and MEK2 kinases-SB203580-a p38 MAPK inhibitor-and SC-514-an IKKβ inhibitor-suppressed cadmium-induced IL-8 expression and release. Upregulation of cadmium-induced IL-6 was inhibited by U0126 and SC-514, but not SB203580. On the other hand, SP600125-a JNK inhibitor-and celecoxib-a selective COX-2 inhibitor-had no effect on production of both cytokines. In conclusion, non-toxic concentrations of cadmium can stimulate IL-6 and IL-8 release through MAPK phosphorylation and NF-κB activation. Suppressing IL-6 and IL-8 production could be novel approaches to prevent cadmium-induced angiogenesis in gliomas and inflammation in the brain., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Treatment with bromelain-containing enzyme preparation for 3-4 weeks is effective for treatment of knee osteoarthritis (OA). Here, we aimed to assess 16-week treatment with bromelain in mild-to-moderate knee OA patients. We performed a randomized, single-blind, active-controlled pilot study. Forty knee OA patients were randomized to receive oral bromelain (500 mg/day) or diclofenac (100 mg/day). Primary outcome was the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) analyzed by Wilcoxon signed rank test. Secondary outcome was the short-form 36 (SF-36). Plasma malondialdehyde (MDA) and nitrite were measured as oxidative stress markers. There was no difference in WOMAC and SF-36 scores compared between bromelain and diclofenac groups after 4 weeks. At week 4, the improvement of total WOMAC and pain subscales from baseline was observed in both groups; however, two patients given diclofenac had adverse effects leading to discontinuation of diclofenac. However, observed treatment difference was inconclusive. At week 16 of bromelain treatment, the patients had improved total WOMAC scores (12.2 versus 25.5), pain subscales (2.4 versus 5.6), stiffness subscales (0.8 versus 2.0), and function subscales (9.1 versus 17.9), and physical component of SF-36 (73.3 versus 65.4) as compared with baseline values. OA patients had higher plasma MDA, nitrite, and prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-stimulated whole blood but lower plasma α-tocopherol than control subjects. Plasma MDA and LPS-stimulated PGE2 production were decreased at week 16 of bromelain treatment. Bromelain has no difference in reducing symptoms of mild-to-moderate knee OA after 4 weeks when compared with diclofenac.

Background: The purpose of this study is to investigate cardiovascular benefits of juices obtained from two commonly consumed fruits in Thailand, Pachyrhizus erosus, L. (yam bean) and Psidium guajava, L. (guava), by examining their acute cardiovascular effects in healthy volunteers. Possible involvements of the dietary nitrate on their effects were investigated as well., Method: Thirty healthy volunteers were randomly divided into three groups of 10 subjects per group and each group was allocated to drink 500 ml of freshly prepared yam bean root juice, guava fruit juice, or water. Systemic nitrate and nitrite concentrations, heart rate, systolic and diastolic blood pressure, serum K(+) concentrations, ex vivo platelet aggregation, and plasma cGMP concentrations were monitored at the baseline and at various time points after the intake of juices or water. Data were compared by repeated measures ANOVA., Results: Following the ingestion of both yam bean root juice and guava fruit juice, collagen-induced but not ADP-induced platelet aggregation was attenuated. Ingestion of yam bean root juice increased systemic nitrate and nitrite concentrations whereby elevated nitrite concentrations correlated with the extent of inhibiting collagen-induced platelet aggregation. In addition, positive correlation between systemic nitrite and plasma cGMP concentrations and negative correlation between plasma cGMP concentrations and the extent of collagen-induced platelet aggregation were revealed. Nevertheless, yam bean root juice reduced only diastolic blood pressure while guava fruit juice reduced heart rate, systolic and diastolic blood pressure., Conclusion: The present study has illustrated, for the first time, acute inhibitory effects of yam bean root juice and guava fruit juice on ex vivo collagen-induced platelet aggregation in healthy subjects. Dietary nitrate was shown to underlie the effect of yam bean root juice but not that of guava fruit juice. Following yam bean root juice ingestion, systemic nitrate apparently converts to nitrite and further to NO which may attenuate platelet responses to collagen stimulation. Cardiovascular benefits of juices from yam bean root and guava fruit are noteworthy in term of the cardiovascular health-promoting approach., Trial Registration: Randomized controlled trial TCTR20150228001 .

Cadmium exposure has been reported to be associated with the risk of vascular disorders. Here, we investigated platelet activity in subjects with chronic cadmium exposure. Eighteen and 15 women participated in this study as chronically cadmium-exposed and control non-exposed subjects, respectively. Plasma P-selectin and CD40 ligand (CD40L), soluble markers of platelet activation, were measured. Platelet aggregation in whole blood, P-selectin and activated glycoprotein (aGP) IIb/IIIa expression on platelets and platelet-leukocyte aggregates were determined. The levels of plasma P-selectin and CD40L increased in subjects with chronic cadmium exposure compared with control subjects. Platelet aggregation induced by adenosine diphosphate (ADP) was higher in cadmium-exposed subjects than control subjects. Cadmium-exposed subjects had higher baseline and ADP-induced aGPIIb/IIIa expression on platelets than control subjects. Platelet-neutrophil aggregates also increased in cadmium-exposed subjects. Blood cadmium correlated with ADP-induced aggregation, aGPIIb/IIIa expression and platelet-neutrophil aggregates, while urinary cadmium correlated with soluble P-selectin. However, cadmium only at high concentration (15 µM) could potentiate ADP-induced platelet activation in vitro. In conclusion, our pilot data show that cadmium-exposed subjects have increased baseline platelet activation and reactivity.

Introduction: Pulmonary arterial hypertension is a life-threatening complication in thalassemia characterized by elevated pulmonary arterial pressure. Increased platelet activation is associated with this complication; however, its role remains unclear., Methods: Platelet activation in splenectomized β-thalassemia/hemoglobin E (Hb E) patients was measured using flow cytometric determination of P-selectin and activated glycoprotein (aGP) IIb/IIIa expression, and platelet-leukocyte aggregates (platelet-neutrophil, platelet-monocyte and platelet-lymphocyte aggregates). Tricuspid regurgitant velocity (TRV) was measured and used as an indicator of pulmonary arterial pressure. Plasma hemoglobin served as markers of hemolysis., Results: Fifteen of 27 patients had elevated TRV (>2.5m/s). Platelet expression of P-selectin and aGPIIb/IIIa, and platelet-leukocyte aggregates were higher in thalassemia patients with elevated TRV than healthy control. Platelet-neutrophil aggregates increased in thalassemia patients with elevated TRV compared to patients with normal TRV. The increase in P-selectin and aGPIIb/IIIa expression induced by adenosine diphosphate (ADP) was higher in patients with elevated TRV than those with normal TRV. Platelet P-selectin expression and platelet-neutrophil aggregates correlated positively with TRV. Plasma hemoglobin levels in patients with elevated TRV were higher than those of the control subjects, and correlated with TRV., Conclusion: Thalassemia patients with elevated TRV have a further increase in platelet activation that correlates with hemolysis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Vascular endothelium is a target of cadmium (Cd) toxicity. Cd exposure has been reported to be associated with vascular disorders. In this study, we aimed to investigate the effects of Cd exposure on markers of endothelial function in human subjects chronically exposed to Cd. Based on blood Cd levels, seventy-five women were categorized into non-exposed, Cd-exposed and severely Cd-exposed groups. Nitrite, L-arginine, asymmetric dimethylarginine (ADMA), and soluble thrombomodulin levels in blood were measured. Nitrite levels were lower in Cd-exposed subjects than non-exposed subjects. Plasma L-arginine decreased while ADMA, an endogenous endothelial nitric oxide synthase (eNOS) inhibitor, increased in Cd-exposed subjects. Soluble thrombomodulin also increased in Cd-exposed subjects. In Cd-exposed subjects, plasma malondialdehyde and protein carbonyl groups increased while the erythrocytic glutathione decreased. Multiple linear regression analysis revealed a negative association between urinary Cd and nitrite levels in erythrocytes. Our research suggests that subjects with chronic Cd exposure have endothelial dysfunction.

Nitric oxide (NO), a small gas molecule, has long been known to be a potent inhibitor of platelet function but the physiological and pathological implications of platelet inhibition by NO have not been well clarified. We recently showed that the addition of nitrite to platelet-rich plasma in the presence of erythrocytes could inhibit platelet aggregation and this inhibitory effect of nitrite + erythrocytes was enhanced by deoxygenation of erythrocytes as measured by P-selectin expression and cGMP production. In order to study the nitrite effect on platelets at different oxygen levels, we used the flow cytometric assays to detect platelet membrane surface markers upon activation. The P-selectin and activated gpIIb/IIIa expression on platelet membranes in response to ADP, collagen and thrombin stimulation was measured at various hematocrit and oxygen levels. Nitrite (0.1 to 1.0 μM) significantly decreased the percentage of these surface markers on the platelet membrane at the hematocrit values above 23% and oxygen levels lower than 49 mmHg. The inhibitory effect of nitrite was augmented by increasing hematocrit values and decreasing oxygen saturation. C-PTIO (an NO scavenger) prevented the platelet inhibition by nitrite + erythrocytes whereas the inhibitors of NO synthase and xanthine oxidoreductase had no effect. These results support the proposal that circulating nitrite decreases platelet reactivity in the presence of partially deoxygenated erythrocytes through its reduction to NO, which may also explain certain differences between arterial and venous thrombosis and support directly the role of deoxyhemoglobin in this process. We believe that our flow cytometric assays offer a possibility to identify the individual molecular process involved in these effects.

Nitrite anion is bioactive nitric oxide (NO) species circulating in blood, and represents the NO bioavailability and endothelial function. In this study, we aimed to investigate the nitrite levels and the correlation with hemolysis and severity in β-thalassemia/hemoglobin E (β-thal/HbE). 38 Children (12.0±1.9 years of age) with a diagnosis of mild, moderate and severe β-thalassemia were enrolled in the study. The blood nitrite levels and potential plasma NO consumption were measured by the chemiluminescence method. The nitrite levels in whole blood and erythrocytes of the severe thalassemia subjects were lower than those of the control subjects. At day 7 after transfusion of packed erythrocytes, the nitrite levels in erythrocytes increased. The plasma hemoglobin and NO consumption increased in the severe thalassemia subjects. The nitrite levels in erythrocytes inversely correlated with plasma hemoglobin, lactate dehydrogenase activity, potential NO consumption, and lipid peroxidation. Our studies demonstrate the decreased NO bioavailability in thalassemia, which could result from endothelial dysfunction, the increased potential NO consumption in plasma by cell-free hemoglobin and oxidative stress., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Potential deleterious effects of red blood cell (RBC) transfusions, especially from blood kept at length, have been ascribed to biochemical changes during storage, including those of nitric oxide (NO) metabolism. Study methods and design: In this study, NO metabolites, nitrite and nitrate, were quantified in RBCs and whole blood with time of storage. Whole blood (WB), leukoreduced (LR), and non-leukoreduced (NLR) components were obtained from healthy volunteer donors and stored in polyvinyl chloride bags for 42 days. Nitrite and nitrate were measured using reductive gas-phase chemiluminescence., Results: Nitrite concentrations initially decreased rapidly from about 150nmol/L, but stabilized at about 44nmol/L in room air for up to 42 days. Nitrate concentrations remained stable during storage at about 35µmol/L. Cells from bags maintained in an argon chamber showed decreased nitrite levels compared to those maintained in room air. Inhibition of enzymes implicated in the NO cycle did not alter nitrite levels., Conclusion: As erythrocytes may contribute to the control of blood flow and oxygen delivery through reduction of nitrite to NO under hypoxic conditions, the present findings provide insight into possible effects of blood transfusion. These measurements may explain some adverse effects of RBC transfusion and suggest ways of optimizing the preservation of stored blood.

Background: Nitrite is a nitric oxide (NO) metabolite in tissues and blood, which can be converted to NO under hypoxia to facilitate tissue perfusion. Although nitrite is known to cause vasodilation following its reduction to NO, the effect of nitrite on platelet activity remains unclear. In this study, the effect of nitrite and nitrite+erythrocytes, with and without deoxygenation, on platelet activity was investigated., Methodology/finding: Platelet aggregation was studied in platelet-rich plasma (PRP) and PRP+erythrocytes by turbidimetric and impedance aggregometry, respectively. In PRP, DEANONOate inhibited platelet aggregation induced by ADP while nitrite had no effect on platelets. In PRP+erythrocytes, the inhibitory effect of DEANONOate on platelets decreased whereas nitrite at physiologic concentration (0.1 µM) inhibited platelet aggregation and ATP release. The effect of nitrite+erythrocytes on platelets was abrogated by C-PTIO (a membrane-impermeable NO scavenger), suggesting an NO-mediated action. Furthermore, deoxygenation enhanced the effect of nitrite as observed from a decrease of P-selectin expression and increase of the cGMP levels in platelets. The ADP-induced platelet aggregation in whole blood showed inverse correlations with the nitrite levels in whole blood and erythrocytes., Conclusion: Nitrite alone at physiological levels has no effect on platelets in plasma. Nitrite in the presence of erythrocytes inhibits platelets through its reduction to NO, which is promoted by deoxygenation. Nitrite may have role in modulating platelet activity in the circulation, especially during hypoxia.

Artemisinin exerts the antimalarial activity through activation by heme. The hemolysis in malaria results in the elevated levels of plasma heme which may affect the activity of artemisinin. We hypothesized that the extracellular heme would potentiate the antimalarial activity of artemisinin. Hemin (ferric heme) at the pathologic concentrations enhanced the activity of artemisinin against Plasmodium falciparum in vitro and increased the levels of the lipid peroxidation products in the presence of artemisinin. The antimalarial activity of artemisinin and potentiation by hemin was decreased by vitamin E. Hemin had no effect on the activity of quinoline drugs (chloroquine, quinine and mefloquine). Furthermore, the oxidative effect of hemin in the presence of artemisinin or quinoline drugs was studied using low-density lipoprotein (LDL) oxidation as a model. Artemisinin enhanced the effects of hemin on lipid peroxidation and a decrease of tryptophan fluorescence in LDL whereas the quinoline drugs inhibited the oxidation by hemin. In conclusion, the extracellular hemin enhances the antimalarial activity of artemisinin as a result of the increasing oxidative effect of hemin.

Increasing evidence indicates that high levels of serum erythropoietin (Epo) can lessen ischemia-reperfusion injury in the heart and multiple cardiac cell types have been suggested to play a role in this Epo effect. To clarify the mechanisms underlying this cardioprotection, we explored Epo treatment of coronary artery endothelial cells and Epo cardioprotection in a Mus musculus model with Epo receptor expression restricted to hematopoietic and endothelial cells (ΔEpoR). Epo stimulation of coronary artery endothelial cells upregulated endothelial nitric oxide synthase (eNOS) activity in vitro and in vivo, and enhanced nitric oxide (NO) production that was determined directly by real-time measurements of gaseous NO release. Epo stimulated phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase kinase (MEK)/extracellular signal regulated kinase (ERK) signaling pathways, and inhibition of PI3K, but not MEK activity, blocked Epo-induced NO production. To verify the potential of this Epo effect in cardioprotection in vivo, ΔEpoR-mice with Epo response in heart restricted to endothelium were treated with Epo. These mice exhibited a similar increase in eNOS phosphorylation in coronary artery endothelium as that found in wild type (WT) mice. In addition, in both WT- and ΔEpoR-mice, exogenous Epo treatment prior to myocardial ischemia provided comparable protection. These data provide the first evidence that endothelial cell response to Epo is sufficient to achieve an acute cardioprotective effect. The immediate response of coronary artery endothelial cells to Epo stimulation by NO production may be a critical mechanism underlying this Epo cardioprotection.

Oxidative modification of low-density lipoprotein (LDL) has been reported in thalassemia, which is a consequence of oxidative stress. However, the levels of oxidized high-density lipoprotein (HDL) in thalassemia have not been evaluated and it is unclear whether HDL oxidation may be linked to LDL oxidation. In this study, the levels of total cholesterol, iron, protein, conjugated diene (CD), lipid hydroperoxide (LOOH), and thiobarbituric acid reactive substances (TBARs) were determined in HDL from healthy volunteers and patients with beta-thalassemia intermedia with hemoglobin E (beta-thal/Hb E). The protective activity of thalassemic HDL on LDL oxidation was also investigated. The iron content of HDL(2) and HDL(3) from beta-thal/HbE patients was higher while the cholesterol content was lower than those in healthy volunteers. Thalassemic HDL(2) and HDL(3) had increased levels of lipid peroxidation markers i.e., conjugated diene, LOOH, and TBARs. Thalassemic HDL had lower peroxidase activity than control HDL and was unable to protect LDL from oxidation induced by CuSO(4). Our findings highlight the oxidative modification and poor protective activity of thalassemic HDL on LDL oxidation which may contribute to cardiovascular complications in thalassemia.

The reaction of deoxyhemoglobin with nitric oxide (NO) or nitrite ions (NO 2 (-)) produces iron-nitrosyl-hemoglobin (HbNO) in contrast to the reaction with oxyhemoglobin, which produces methemoglobin and nitrate (NO 3 (-)). HbNO has not been associated with the known bioactivities of NO. We hypothesized that HbNO in erythrocytes could be an important source of bioactive NO/nitrite if its oxidation was coupled to the ascorbic acid (ASC) cycle. Studied by absorption and electron paramagnetic resonance (EPR) spectroscopy, DHA oxidized HbNO to methemoglobin and liberated NO from HbNO as determined by chemiluminescence. Both DHA and ascorbate free radical (AFR), the intermediate between ASC and DHA, enhanced NO oxidation to nitrite, but not nitrate; nor did either oxidize nitrite to nitrate. DHA increased the basal levels of nitrite in erythrocytes, while the reactions of nitrite with hemoglobin are slow. In erythrocytes loaded with HbNO, HbNO disappeared after DHA addition, and the AFR signal was detected by EPR. We suggest that the ASC-AFR-DHA cycle may be coupled to that of HbNO-nitrite and provide a mechanism for the endocrine transport of NO via hemoglobin within erythrocytes, resulting in the production of intracellular nitrite. Additionally, intracellular nitrite and nitrate seem to be largely generated by independent pathways within the erythrocyte. These data provide a physiologically robust mechanism for erythrocytic transport of NO bioactivity allowing for hormone-like properties.

Introduction: Oxidative stress has been demonstrated in malaria. The potential oxidative modification of lipoproteins derived from malaria patients was studied. These oxidized lipids may have role in pathogenesis of malaria., Method: The plasma lipid profile and existence of oxidized forms of very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) were investigated in malaria (17 mild and 24 severe patients) and 37 control subjects. Thiobarbituric acid reactive substances (TBARs), conjugated dienes, tryptophan fluorescence and fluidity of lipoproteins were determined as markers of oxidation. The biological effect of malarial lipoproteins was assessed by the expression of adhesion molecules on endothelial cells., Results: Malarial lipoproteins had decreased cholesterol (except in VLDL) and phospholipid. The triglyceride levels were unchanged. The cholesterol/phospholipid ratio of LDL was decreased in malaria, but increased in VLDL and HDL. TBARs and conjugate dienes were increased in malarial lipoproteins, while the tryptophan fluorescence was decreased. The fluidity of lipoproteins was increased in malaria. These indicated the presence of oxidized lipoproteins in malaria by which the degree of oxidation was correlated with severity. Of three lipoproteins from malarial patients, LDL displayed the most pronounced oxidative modification. In addition, oxidized LDL from malaria patients increased endothelial expression of adhesion molecules., Conclusion: In malaria, the lipoproteins are oxidatively modified, and the degree of oxidation is related with severity. Oxidized LDL from malarial patients increases the endothelial expression of adhesion molecules. These suggest the role of oxidized lipoproteins, especially LDL, on the pathogenesis of disease.

Artemisinin, a sesquiterpene with endoperoxide bond, possesses potent antimalarial activity against the ring and late stage of chloroqine-resistant Plasmodium falciparum malaria both in vitro and in vivo. The mode of antimalarial activity of artemisinin is iron-dependent. The aim of this study was to investigate the reactions of artemisinin with ferrous and ferric ions in aqueous buffer. Artemisinin generated a cycle of iron oxidation and reduction. It oxidized ferrous and reduced ferric ions with similar rate of reaction (k=10+/-0.5 M(-1) x s(-1) for ferrous and k=8.5+/-2.0 M(-1) x s(-1) for ferric ion). The major active product was dihydroartemisinin which exhibited antimalarial activity at least 3 times more potent than artemisinin. Dihydroartemisinin preferably binds to ferric ion, forming ferric-dihydroartemisinin complex. The re-oxidation of the complex gives artemisinin and ferric ion. This suggests that in aqueous buffer, the reaction of artemisinin with iron may give rise to the active reaction products, one of them being dihydroartemisinin, which is responsible for antimalarial activity.

The effect of artemisinin on membrane fluidity of erythrocytes was investigated using spin labeling compounds, doxyl stearic acids. The membrane fluidity of erythrocytes from the in vitro culture and malaria patients was determined. In vitro, the erythrocytes in parasite culture showed an increase in membrane fluidity which was associated with the parasite counts and stage of parasites. Artemisinin caused reduction in membrane fluidity and the effect was more pronounced in the erythrocytes infected with schizont stage-parasites. In vivo, the elevation of plasma TBARs (thiobarbituric acid reactive substances) and reduction of membrane fluidity were evident in Plasmodium falciparum-infected patients, particularly in severe cases. The levels of plasma TBARs were related to the severity of the disease. Treatment with artemisinin alone showed no effect on plasma TBARs, and did not alter the membrane fluidity. Desferrioxamine, however, reduced oxidative damage during the infection without compromising the therapeutic effect of artemisinin. These findings suggested that the infected erythrocytes were prone to the effect of artemisinin. Addition of a chelator such as desferrioxamine is beneficial and can improve the treatment of severe malaria.