Iron scavenging and myeloid cell polarization.

Myeloid cells that populate all human organs and blood are a versatile class of innate immune cells. They are crucial for sensing and regulating processes as diverse as tissue homeostasis and inflammation and are frequently characterized by their roles in either regulating or promoting inflammation. Recent studies in cultured cells and mouse models highlight the role of iron in skewing the functional properties of myeloid cells in tissue damage and repair. Here, we review certain emerging concepts on how iron influences and determines myeloid cell polarization in the context of its uptake, storage, and metabolism, including in conditions such as multiple sclerosis (MS), sickle cell disease, and tumors.

Heme-induced loss of renovascular endothelial protein C receptor promotes chronic kidney disease in sickle mice.

ABSTRACT: Chronic kidney disease (CKD) is a major contributor to morbidity and mortality in sickle cell disease (SCD). Anemia, induced by chronic persistent hemolysis, is associated with the progressive deterioration of renal health, resulting in CKD. Moreover, patients with SCD experience acute kidney injury (AKI), a risk factor for CKD, often during vaso-occlusive crisis associated with acute intravascular hemolysis. However, the mechanisms of hemolysis-driven pathogenesis of the AKI-to-CKD transition in SCD remain elusive. Here, we investigated the role of increased renovascular rarefaction and the resulting substantial loss of the vascular endothelial protein C receptor (EPCR) in the progressive deterioration of renal function in transgenic SCD mice. Multiple hemolytic events raised circulating levels of soluble EPCR (sEPCR), indicating loss of EPCR from the cell surface. Using bone marrow transplantation and super-resolution ultrasound imaging, we demonstrated that SCD mice overexpressing EPCR were protective against heme-induced CKD development. In a cohort of patients with SCD, plasma sEPCR was significantly higher in individuals with CKD than in those without CKD. This study concludes that multiple hemolytic events may trigger CKD in SCD through the gradual loss of renovascular EPCR. Thus, the restoration of EPCR may be a therapeutic target, and plasma sEPCR can be developed as a prognostic marker for sickle CKD.

Hemoglobin scavenger receptor CD163 as a potential biomarker of hemolysis-induced hepatobiliary injury in sickle cell disease.

Sickle cell disease (SCD)-associated chronic hemolysis promotes oxidative stress, inflammation, and thrombosis leading to organ damage, including liver damage. Hemoglobin scavenger receptor CD163 plays a protective role in SCD by scavenging both hemoglobin-haptoglobin complexes and cell-free hemoglobin. A limited number of studies in the past have shown a positive correlation of CD163 expression with poor disease outcomes in patients with SCD. However, the role and regulation of CD163 in SCD-related hepatobiliary injury have not been fully elucidated yet. Here we show that chronic liver injury in SCD patients is associated with elevated levels of hepatic membrane-bound CD163. Hemolysis and increase in hepatic heme, hemoglobin, and iron levels elevate CD163 expression in the SCD mouse liver. Mechanistically we show that heme oxygenase-1 (HO-1) positively regulates membrane-bound CD163 expression independent of nuclear factor erythroid 2-related factor 2 (NRF2) signaling in SCD liver. We further demonstrate that the interaction between CD163 and HO-1 is not dependent on CD163-hemoglobin binding. These findings indicate that CD163 is a potential biomarker of SCD-associated hepatobiliary injury. Understanding the role of HO-1 in membrane-bound CD163 regulation may help identify novel therapeutic targets for hemolysis-induced chronic liver injury.

Nitric Oxide Resistance in Priapism Associated with Sickle Cell Disease: Mechanisms, Therapeutic Challenges, and Future Directions.

Patients with sickle cell disease (SCD) display priapism, a prolonged penile erection in the absence of sexual arousal. The current pharmacological treatments for SCD-associated priapism are limited and focused on acute interventions rather than prevention. Thus, there is an urgent need for new drug targets and preventive pharmacological therapies for this condition. This review focuses on the molecular mechanisms linked to the dysfunction of the NO-cyclic guanosine monophosphate (cGMP)-phosphodiesterase type 5 (PDE5) pathway implicated in SCD-associated priapism. In murine models of SCD, reduced nitric oxide (NO)-cGMP bioavailability in the corpus cavernosum is associated with elevated plasma hemoglobin levels, increased reactive oxygen species levels that inactive NO, and testosterone deficiency that leads to endothelial nitric oxide synthase downregulation. We discuss the consequences of the reduced cGMP-dependent PDE5 activity in response to these molecular changes, highlighting it as the primary pathophysiological mechanism leading to excessive corpus cavernosum relaxation, culminating in priapism. We also further discuss the impact of intravascular hemolysis on therapeutic approaches, present current pharmacological strategies targeting the NO-cGMP-PDE5 pathway in the penis, and identify potential pharmacological targets for future priapism therapies. In men with SCD and priapism, PDE5 inhibitor therapy and testosterone replacement have shown promising results. Recent preclinical research reported the beneficial effect of treatment with haptoglobin and NO donors. SIGNIFICANCE STATEMENT: This review discusses the molecular changes that reduce NO-cGMP bioavailability in the penis in SCD and highlights pharmacological targets and therapeutic strategies for the treatment of priapism, including PDE5 inhibitors, hormonal modulators, NO donors, hydroxyurea, soluble guanylate cyclase stimulators, haptoglobin, hemopexin, and antioxidants.

Effect of voxelotor on murine bone marrow and peripheral blood with hematopoietic progenitor cell mobilization for gene therapy of sickle cell disease.

In preparation for hematopoietic stem cell mobilization and collection, current ex vivo gene therapy protocols for sickle cell disease require patients to undergo several months of chronic red cell transfusion. For health care equity, alternatives to red cell transfusion should be available. We examined whether treatment with GBT1118, the murine analog of voxelotor, could be a safe and feasible alternative to red cell transfusion. We found that 3 weeks of treatment with GBT1118 increased the percentage of bone marrow hematopoietic stem cells and upon plerixafor mobilization, the percentage of peripheral blood hematopoietic stem cells. Our data suggest that voxelotor should be further explored for its potential safety and utility as preparation for hematopoietic stem cell mobilization and collection.

Bach1 inhibitor HPP-D mediates γ-globin gene activation in sickle erythroid progenitors.

Sickle cell disease (SCD) is the most common ß-hemoglobinopathy caused by various mutations in the adult ß-globin gene resulting in sickle hemoglobin production, chronic hemolytic anemia, pain, and progressive organ damage. The best therapeutic strategies to manage the clinical symptoms of SCD is the induction of fetal hemoglobin (HbF) using chemical agents. At present, among the Food and Drug Administration-approved drugs to treat SCD, hydroxyurea is the only one proven to induce HbF protein synthesis, however, it is not effective in all people. Therefore, we evaluated the ability of the novel Bach1 inhibitor, HPP-D to induce HbF in KU812 cells and primary sickle erythroid progenitors. HPP-D increased HbF and decreased Bach1 protein levels in both cell types. Furthermore, chromatin immunoprecipitation assay showed reduced Bach1 and increased NRF2 binding to the γ-globin promoter antioxidant response elements. We also observed increased levels of the active histone marks H3K4Me1 and H3K4Me3 supporting an open chromatin configuration. In primary sickle erythroid progenitors, HPP-D increased γ-globin transcription and HbF positive cells and reduced sickled erythroid progenitors under hypoxia conditions. Collectively, our data demonstrate that HPP-D induces γ-globin gene transcription through Bach1 inhibition and enhanced NRF2 binding in the γ-globin promoter antioxidant response elements.

Endurance training improves oxygen uptake/demand mismatch, metabolic flexibility and recovery in patients with sickle cell disease.

Patients with sickle cell disease (SCD) display lower slope coefficients of the oxygen uptake (V̇O2) versus work rate (W) relationship (delineating an O2 uptake/demand mismatch) and a poor metabolic flexibility. Because endurance training improves the microvascular network and increases the activity of oxidative enzymes, including one involved in lipid oxidation, endurance training might improve the slope coefficient of the V̇O2 versus W curve and the metabolic flexibility of SCD patients. Endurance training may also contribute to improve patients' post-exercise cardiopulmonary and metabolic recovery. Fifteen patients with SCD performed a submaximal incremental test on a cycle ergometer before (SIT1) and after (SIT2) 8 weeks of endurance training. Minute ventilation (V̇ E), ventilation rate, heart rate, V̇O2, carbon dioxide production (V̇CO2), respiratory exchange ratio, carbohydrate/lipid utilization and partitioning (including %Lipidox) and blood lactate concentration were measured during and after SIT1 and SIT2. At baseline, the slope coefficient of the V̇O2 versus W curve positively correlated with total hemoglobin, mean corpuscular hemoglobin and percentage of HbF. After training, the slope coefficient of the V̇O2 versus W curve was significantly higher and the increase in blood lactate concentration was delayed. If patients' energy metabolism apparently relied largely on carbohydrate sources during SIT1, %Lipidox tended to increase at low exercise intensities during SIT2, supporting a training-induced improvement of metabolic flexibility in patients with SCD. Post-exercise recovery of ventilation rate, V̇ E/V̇CO2, heart rate and blood lactate concentration was faster after training. We concluded that exercise training in patients with SCD: (i) ameliorated the oxygen uptake/ demand mismatch, (ii) blunted the metabolic inflexibility, and (iii) improved post-exercise cardiopulmonary and metabolic responses.

Epeleuton, a novel synthetic ω-3 fatty acid, reduces hypoxia/ reperfusion stress in a mouse model of sickle cell disease.

Inflammatory vasculopathy is critical in sickle cell disease (SCD)-associated organ damage. An imbalance between pro-inflammatory and pro-resolving mechanisms in response to different triggers such as hypoxia/reoxygenation or infections has been proposed to contribute to the progression of SCD. Administration of specialized pro-resolving lipid mediators may provide an effective therapeutic strategy to target inflammatory vasculopathy and to modulate inflammatory response. Epeleuton (15 hydroxy eicosapentaenoic acid ethyl ester) is a novel, orally administered, second-generation ω-3 fatty acid with a favorable clinical safety profile. In this study we show that epeleuton re-programs the lipidomic pattern of target organs for SCD towards a pro-resolving pattern. This protects against systemic and local inflammatory responses and improves red cell features, resulting in reduced hemolysis and sickling compared with that in vehicle-treated SCD mice. In addition, epeleuton prevents hypoxia/reoxygenation-induced activation of nuclear factor-κB with downregulation of the NLRP3 inflammasome in lung, kidney, and liver. This was associated with downregulation of markers of vascular activation in epeleuton-treated SCD mice when compared to vehicle-treated animals. Collectively our data support the potential therapeutic utility of epeleuton and provide the rationale for the design of clinical trials to evaluate the efficacy of epeleuton in patients with SCD.

Endothelial dysfunction in Sickle Cell Disease: Strategies for the treatment.

Sickle Cell Anemia (SCA), is an inherited hemoglobinopathy characterized by the presence of an abnormal hemoglobin (HbS), being the most prevalent sickle cell disease (SCD). SCA is characterized by vascular endothelial dysfunction, which contributes significantly to various clinical conditions, including but not limited to pulmonary hypertension, priapism, cutaneous leg ulceration, and stroke. The pathophysiology of endothelial dysfunction (ED) in SCA is a multifaceted process involving a chronic inflammatory and hypercoagulable state. Key factors include hemolysis-associated elements like reduced arginine and nitric oxide (NO) availability, elevated levels of vascular adhesion molecules, the uncoupling effect of NO synthase, heightened arginase activity, an environment characterized by oxidative stress with the production of reactive oxygen and nitrogen species, and occurrences of ischemia-reperfusion injury, along with apolipoprotein A-1 depletion. The urgency for novel interventions addressing ED is evident. Presently, there is a focus on investigating small molecules that disrupt the arginine-nitric oxide pathway, exhibiting anti-inflammatory and antioxidant properties while diminishing levels of cellular and vascular adhesion molecules. In this mini-review article, we delve into the progress made in strategies for treating ED in SCD with the aim of cultivating insights for drug design.

Doping control approach: Identification of equine in vitro metabolites of voxelotor (GBT440), a hemoglobin S polymerization inhibitor.

RATIONALE: Sickle cell disease, a debilitating genetic disorder affecting numerous newborns globally, has historically received limited attention in pharmaceutical research. However, recent years have witnessed a notable shift, with the Food and Drug Administration approving three innovative disease-modifying medications. Voxelotor, also known as GBT440, is a promising compound that effectively prevents sickling, providing a safe approach to alleviate chronic hemolytic anemia in sickle cell disease. It is a novel, orally bioavailable small molecule that inhibits hemoglobin S polymerization by enhancing oxygen affinity to hemoglobin. The investigation demonstrated that voxelotor led to an unintended elevation of hemoglobin levels in healthy individuals by increasing serum erythropoietin levels. METHODS: Voxelotor and its metabolites in an in vitro setting utilizing equine liver microsomes were discussed. Plausible structures of the identified metabolites were inferred through the application of liquid chromatography in conjunction with high-resolution mass spectrometry. RESULTS: Under the experimental conditions, a total of 31 metabolites were detected, including 16 phase I metabolites, two phase II metabolites, and 13 conjugates of phase I metabolites. The principal phase I metabolites were generated through processes such as hydroxylation, reduction, and dissociation. The presence of glucuronide and sulfate conjugates of the parent drug were also observed, along with hydroxylated, reduced, and dissociated analogs. CONCLUSIONS: The data acquired will accelerate the identification of voxelotor and related compounds, aiding in the detection of their illicit use in competitive sports. It is crucial to emphasize that the metabolites detailed in this manuscript were identified through in vitro experiments and their detection in an in vivo study may not be guaranteed.

Transcriptional Repressor BCL11A in Erythroid Cells.

BCL11A, a zinc finger repressor, is a stage-specific transcription factor that controls the switch from fetal (HbF, α2γ2) to adult (HbA, α2ß2) hemoglobin in erythroid cells. While BCL11A was known as a factor critical for B-lymphoid cell development, its relationship to erythroid cells and HbF arose through genome-wide association studies (GWAS). Subsequent work validated its role as a silencer of γ-globin gene expression in cultured cells and mice. Erythroid-specific loss of BCL11A rescues the phenotype of engineered sickle cell disease (SCD) mice, thereby suggesting that downregulation of BCL11A expression might be beneficial in patients with SCD and ß-thalassemia. Common genetic variation in GWAS resides in an erythroid-specific enhancer within the BCL11A gene that is required for its own expression. CRISPR/Cas9 gene editing of the enhancer revealed a GATA-binding site that confers a large portion of its regulatory function. Disruption of the GATA site leads to robust HbF reactivation. Advancement of a guide RNA targeting the GATA-binding site in clinical trials has recently led to approval of first-in-man use of ex vivo CRISPR editing of hematopoietic stem/progenitor cells (HSPCs) as therapy of SCD and ß-thalassemia. Future challenges include expanding access and infrastructure for delivery of genetic therapy to eligible patients, reducing potential toxicity and costs, exploring prospects for in vivo targeting of hematopoietic stem cells (HSCs), and developing small molecule drugs that impair function of BCL11A protein as an alternative option.

Metabolic regulation of erythrocyte development and disorders.

The formation of new red blood cells (RBC) (erythropoiesis) has served as a paradigm for understanding cellular differentiation and developmental control of gene expression. The metabolic regulation of this complex, coordinated process remains poorly understood. Each step of erythropoiesis, including lineage specification of hematopoietic stem cells, proliferation, differentiation, and terminal maturation into highly specialized oxygen-carrying cells, has unique metabolic requirements. Developing erythrocytes in mammals are also characterized by unique metabolic events such as loss of mitochondria with switch to glycolysis, ejection of nucleus and organelles, high-level heme and hemoglobin synthesis, and antioxidant requirement to protect hemoglobin molecules. Genetic defects in metabolic enzymes, including pyruvate kinase and glucose-6-phosphate dehydrogenase, cause common erythrocyte disorders, whereas other inherited disorders such as sickle cell disease and ß-thalassemia display metabolic abnormalities associated with disease pathophysiology. Here we describe recent discoveries on the metabolic control of RBC formation and function, highlight emerging concepts in understanding the erythroid metabolome, and discuss potential therapeutic benefits of targeting metabolism for RBC disorders.

Biased agonism of protease-activated receptor-1 regulates thromboinflammation in murine sickle cell disease.

ABSTRACT: Sickle cell disease (SCD) is a hereditary hemoglobinopathy marked by hemolytic anemia and vaso-occlusive events (VOEs). Chronic endothelial activation, inflammation, and coagulation activation contribute to vascular congestion, VOEs, and end-organ damage. Coagulation proteases such as thrombin and activated protein C (APC) modulate inflammation and endothelial dysfunction by activating protease-activated receptor 1 (PAR1), a G-protein-coupled receptor. Thrombin cleaves PAR1 at Arg41, while APC cleaves PAR1 at Arg46, initiating either proinflammatory or cytoprotective signaling, respectively, a signaling conundrum known as biased agonism. Our prior research established the role of thrombin and PAR1 in vascular stasis in an SCD mouse model. However, the role of APC and APC-biased PAR1 signaling in thrombin generation, inflammation, and endothelial activation in SCD remains unexplored. Inhibition of APC in SCD mice increased thrombin generation, inflammation, and endothelial activation during both steady state and tumor necrosis factor α challenge. To dissect the individual contributions of thrombin-PAR1 and APC-PAR1 signaling, we used transgenic mice with point mutations at 2 PAR1 cleavage sites, ArgR41Gln (R41Q) imparting insensitivity to thrombin and Arg46Gln (R46Q) imparting insensitivity to APC. Sickle bone marrow chimeras expressing PAR1-R41Q exhibited reduced thrombo-inflammatory responses compared with wild type PAR1 or PAR1-R46Q mice. These findings highlight the potential benefit of reducing thrombin-dependent PAR1 activation while preserving APC-PAR1 signaling in SCD thromboinflammation. These results also suggest that pharmacological strategies promoting biased PAR1 signaling could effectively mitigate vascular complications associated with SCD.

Impacts of oxidative stress and anti-oxidants on the development, pathogenesis, and therapy of sickle cell disease: A comprehensive review.

Sickle cell disease (SCD) is the most severe monogenic hemoglobinopathy caused by a single genetic mutation that leads to repeated polymerization and depolymerization of hemoglobin resulting in intravascular hemolysis, cell adhesion, vascular occlusion, and ischemia-reperfusion injury. Hemolysis causes oxidative damage indirectly by generating reactive oxygen species through various pathophysiological mechanisms, which include hemoglobin autoxidation, endothelial nitric oxide synthase uncoupling, reduced nitric oxide bioavailability, and elevated levels of asymmetric dimethylarginine. Red blood cells have a built-in anti-oxidant system that includes enzymes like sodium dismutase, catalase, and glutathione peroxidase, along with free radical scavenging molecules, such as vitamin C, vitamin E, and glutathione, which help them to fight oxidative damage. However, these anti-oxidants may not be sufficient to prevent the effects of oxidative stress in SCD patients. Therefore, in line with a recent FDA request that the focus to be placed on the development of innovative therapies for SCD that address the root cause of the disease, there is a need for therapies that target oxidative stress and restore redox balance in SCD patients. This review summarizes the current state of knowledge regarding the role of oxidative stress in SCD and the potential benefits of anti-oxidant therapies. It also discusses the challenges and limitations of these therapies and suggests future directions for research and development.

Unraveling DEHP influence on hemoglobin S polymerization in sickle cell disease: Ex vivo, in vitro and in silico analysis.

Sickle cell disease (SCD) is a hereditary hemoglobinopathy, caused by a mutation at position 6 of the ß-globin chain and patients are frequently exposed to several blood transfusions in order to maintain physiological function. Transfusion blood bags are composed of PVC and phthalates (as DEHP) are often introduced to the material in order to confer malleability. In this sense, DEHP can easily elute to the blood and cause harmful effects. This study aimed to unravel DEHP effect on SCD patient's hemoglobin function. We found that HbS polymerization using whole erythrocytes is decreased by DEHP in ex vivo experiments and this effect might be mediated by the DEHP-VAL6 interaction, evaluated in silico. Isolated HbS exhibited less polymerization at low DEHP concentrations and increased polymerization rate at higher concentration. When analyzing the propensity to aggregate, HbS is more inclined to aggregate when compared to HbA due to the residue 6 mutation. Circular dichroism showed characteristic hemoglobin peaks for oxygenated HbS that are lost when oxygen is sequestered, and DEHP at higher concentration mildly recovers a peak close to the second hemoglobin one. Finally, by transmission electron microscopy we demonstrated that high DEHP concentration increased polymer formation with a more organized structure. These findings show for the first-time the beneficial effect of low-dose DEHP on HbS polymerization.

Comparison of cerebral oxygen extraction fraction using ASE and TRUST methods in patients with sickle cell disease and healthy controls.

Abnormal oxygen extraction fraction (OEF), a putative biomarker of cerebral metabolic stress, may indicate compromised oxygen delivery and ischemic vulnerability in patients with sickle cell disease (SCD). Elevated OEF was observed at the tissue level across the brain using an asymmetric spin echo (ASE) MR method, while variable global OEFs were found from the superior sagittal sinus (SSS) using a T2-relaxation-under-spin-tagging (TRUST) MRI method with different calibration models. In this study, we aimed to compare the average ASE-OEF in the SSS drainage territory and TRUST-OEF in the SSS from the same SCD patients and healthy controls. 74 participants (SCD: N = 49; controls: N = 25) underwent brain MRI. TRUST-OEF was quantified using the Lu-bovine, Bush-HbA and Li-Bush-HbS models. ASE-OEF and TRUST-OEF were significantly associated in healthy controls after controlling for hematocrit using the Lu-bovine or the Bush-HbA model. However, no association was found between ASE-OEF and TRUST-OEF in patients with SCD using either the Bush-HbA or the Li-Bush-HbS model. Plausible explanations include a discordance between spatially volume-averaged oxygenation brain tissue and flow-weighted volume-averaged oxygenation in SSS or sub-optimal calibration in SCD. Further work is needed to refine and validate non-invasive MR OEF measurements in SCD.

PGC-1α agonism induces fetal hemoglobin and exerts antisickling effects in sickle cell disease.

Sickle cell disease is a growing health burden afflicting millions around the world. Clinical observation and laboratory studies have shown that the severity of sickle cell disease is ameliorated in individuals who have elevated levels of fetal hemoglobin. Additional pharmacologic agents to induce sufficient fetal hemoglobin to diminish clinical severity is an unmet medical need. We recently found that up-regulation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) can induce fetal hemoglobin synthesis in human primary erythroblasts. Here, we report that a small molecule, SR-18292, increases PGC-1α leading to enhanced fetal hemoglobin expression in human erythroid cells, ß-globin yeast artificial chromosome mice, and sickle cell disease mice. In SR-18292-treated sickle mice, sickled red blood cells are significantly reduced, and disease complications are alleviated. SR-18292, or agents in its class, could be a promising additional therapeutic for sickle cell disease.

A molecular glue degrader of the WIZ transcription factor for fetal hemoglobin induction.

Sickle cell disease (SCD) is a prevalent, life-threatening condition attributable to a heritable mutation in ß-hemoglobin. Therapeutic induction of fetal hemoglobin (HbF) can ameliorate disease complications and has been intently pursued. However, safe and effective small-molecule inducers of HbF remain elusive. We report the discovery of dWIZ-1 and dWIZ-2, molecular glue degraders of the WIZ transcription factor that robustly induce HbF in erythroblasts. Phenotypic screening of a cereblon (CRBN)-biased chemical library revealed WIZ as a previously unknown repressor of HbF. WIZ degradation is mediated by recruitment of WIZ(ZF7) to CRBN by dWIZ-1, as resolved by crystallography of the ternary complex. Pharmacological degradation of WIZ was well tolerated and induced HbF in humanized mice and cynomolgus monkeys. These findings establish WIZ degradation as a globally accessible therapeutic strategy for SCD.

Effects of oxidative stress on liver, brain and spinal cord of rats using L-NAME and treated with hydroxyurea. A model of sickle cell complication

Abstract Purpose: To analyze the serum levels of nitric oxide and correlate them with the levels of thiobarbituric acid reactive substances (TBARS) in liver, brain and spinal cord of animals using L-NAME and treated with hydroxyurea. Methods: Eighteen male albino Wistar rats were divided into three groups. NG-nitro-L-arginine methyl ester (L-NAME) was intraperitoneally administered to induce oxidative stress. TBARS and plasma nitric oxide levels were analyzed in all groups. Histopathology of the liver and vascular tissue was performed. Results: Statistically significant differences were seen in liver, brain and spinal cord TBARS levels. Conclusions: Following the use of L-NAME, hepatic tissue increased the number of Kupffer cells as oxidative stress and inflammatory response increased. The use of L-NAME caused an increase in lipid peroxidation products and, consequently, in oxidative stress in animals. Hydroxyurea doses of 35 mg / kg / day reduced TBARS values in liver, brain and spinal cord.