Curcuminoids supplementation ameliorates iron overload, oxidative stress, hypercoagulability, and inflammation in non-transfusion-dependent ß-thalassemia/Hb E patients.

Curcuminoids, polyphenol compounds in turmeric, possess several pharmacological properties including antioxidant, iron-chelating, and anti-inflammatory activities. Effects of curcuminoids in thalassemia patients have been explored in a limited number of studies using different doses of curcuminoids. The present study aims to evaluate the effects of 24-week curcuminoids supplementation at the dosage of 500 and 1000 mg/day on iron overload, oxidative stress, hypercoagulability, and inflammation in non-transfused ß-thalassemia/Hb E patients. In general, both curcuminoids dosages significantly lowered the levels of oxidative stress, hypercoagulability, and inflammatory markers in the patients. In contrast, reductions in iron parameter levels were more remarkable in the 1000 mg/day group. Subgroup analysis revealed that a marker of hypercoagulability was significantly decreased only in patients with baseline ferritin ≤ 1000 ng/ml independently of curcuminoids dosage. Moreover, the alleviation of iron loading parameters was more remarkable in patients with baseline ferritin > 1000 ng/ml who receive 1000 mg/day curcuminoids. On the other hand, the responses of oxidative stress markers were higher with 500 mg/day curcuminoids regardless of baseline ferritin levels. Our study suggests that baseline ferritin levels should be considered in the supplementation of curcuminoids and the appropriate curcuminoids dosage might differ according to the required therapeutic effect. Thai Clinical Trials Registry (TCTR): TCTR20200731003; July 31, 2020 "retrospectively registered".

Differential plasma proteome profiles of mild versus severe ß-thalassemia/Hb E.

The severity of thalassemia is currently classified based on clinical manifestations and multiple tests. In the present study, we performed a plasma proteome analysis to identify differentially expressed proteins compared between normal subjects and patients with mild and severe forms of ß-thalassemia/hemoglobin E (Hb E). Plasma samples were collected from patients with mild (n = 8) and severe (n = 12) forms as well as healthy normal individuals (n = 12). Clinical chemistry revealed that several parameters, i.e., hematological indices, oxidative stress markers, antioxidant enzymes, and erythropoietic activity, had significant differences among these three groups. After removal of seven major abundant proteins, the plasma proteome profiles were compared using two-dimensional gel electrophoresis. Spot matching, quantitative intensity analysis, and statistics revealed differential levels of 32 and 9 proteins when comparing normal vs. patients and mild vs. severe forms, respectively. These proteins were successfully identified by quadrupole time-of-flight mass spectrometry and/or tandem mass spectrometry. The decreased level of ADP-ribosylation factor guanine nucleotide-exchange factor 2 in ß-thalassemia/Hb E patients compared to healthy individuals and the decreased level of endothelin-converting enzyme 2 in severe form compared to the mild form of the disease were validated by Western blot analysis. Our data provide a number of proteins that may lead to better understanding of the pathophysiology of thalassemia or for novel biomarkers which can be used to simply differentiate mild and severe forms of ß-thalassemia/Hb E without any need for multiple tests.

Glutathione redox system in ß -thalassemia/Hb E patients.

ß -thalassemia/Hb E is known to cause oxidative stress induced by iron overload. The glutathione system is the major endogenous antioxidant that protects animal cells from oxidative damage. This study aimed to determine the effect of disease state and splenectomy on redox status expressed by whole blood glutathione (GSH)/glutathione disulfide (GSSG) and also to evaluate glutathione-related responses to oxidation in ß -thalassemia/Hb E patients. Twenty-seven normal subjects and 25 ß -thalassemia/Hb E patients were recruited and blood was collected. The GSH/GSSG ratio, activities of glutathione-related enzymes, hematological parameters, and serum ferritin levels were determined in individuals. Patients had high iron-induced oxidative stress, shown as significantly increased serum ferritin, a decreased GSH/GSSG ratio, and increased activities of glutathione-related enzymes. Splenectomy increased serum ferritin levels and decreased GSH levels concomitant with unchanged glutathione-related enzyme activities. The redox ratio had a positive correlation with hemoglobin levels and negative correlation with levels of serum ferritin. The glutathione system may be the body's first-line defense used against oxidative stress and to maintain redox homeostasis in thalassemic patients based on the significant correlations between the GSH/GSSH ratio and degree of anemia or body iron stores.

Xanthones from mangosteen inhibit inflammation in human macrophages and in human adipocytes exposed to macrophage-conditioned media.

Obesity-associated inflammation is characterized by recruitment of macrophages (MPhi) into white adipose tissue (WAT) and production of inflammatory cytokines, leading to the development of insulin resistance. The xanthones, alpha- and gamma-mangostin (MG), are major bioactive compounds found in mangosteen that are reported to have antiinflammatory and antioxidant properties. Thus, we examined the efficacy of MG to prevent lipopolysaccharide (LPS)-mediated inflammation in human MPhi (differentiated U937 cells) and cross-talk with primary cultures of newly differentiated human adipocytes. We found that alpha- and gamma-MG attenuated LPS-induced expression of inflammatory genes, including tumor necrosis factor-alpha, interleukin-6, and interferon gamma-inducible protein-10 in a dose-dependent manner in MPhi. We also found that alpha- and gamma-MG attenuated LPS-activated mitogen-activated protein kinases (MAPK) and activator protein (AP)-1, but only gamma-MG reduced nuclear factor-kappaB (NF-kappaB). In addition, alpha- and gamma-MG attenuated LPS suppression of PPARgamma gene expression in a dose-dependent manner. Notably, the ability of MPhi-conditioned media to cause inflammation and insulin resistance in primary cultures of human adipocytes was attenuated by pretreating MPhi with gamma-MG. Taken together, these data demonstrate that MG attenuates LPS-mediated inflammation in MPhi and insulin resistance in adipocytes, possibly by preventing the activation of MAPK, NF-kappaB, and AP-1, which are central to inflammatory cytokine production in WAT.

Xanthones from mangosteen prevent lipopolysaccharide-mediated inflammation and insulin resistance in primary cultures of human adipocytes.

The xanthones, alpha- and gamma-mangostin (MG), are major bioactive compounds found in mangosteen and are reported to have antiinflammatory properties in several murine models. Given the association between obesity, chronic low-grade inflammation, and insulin resistance, we examined the effects of alpha- and gamma-MG on markers of inflammation and insulin resistance in primary cultures of newly differentiated human adipocytes treated with lipopolysaccharide (LPS). alpha- and gamma-MG decreased the induction by LPS of inflammatory genes, including tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, IL-8, monocyte chemoattractant protein-1, and Toll-like receptor-2. Moreover, alpha- and gamma-MG attenuated LPS activation of the mitogen-activated protein kinases (MAPK) c-jun NH(2)-terminal kinase, extracellular signal-related kinase, and p38. alpha- and gamma-MG also attenuated LPS activation of c-Jun and activator protein (AP)-1 activity. gamma-MG was more effective than alpha-MG on an equimolar basis. Furthermore, gamma-MG but not alpha-MG attenuated LPS-mediated IkappaB-alpha degradation and nuclear factor-kappaB (NF-kappaB) activity. In addition, gamma-MG prevented the suppression by LPS of insulin-stimulated glucose uptake and PPAR-gamma and adiponectin gene expression. Taken together, these data demonstrate that MG attenuates LPS-mediated inflammation and insulin resistance in human adipocytes, possibly by inhibiting the activation of MAPK, NF-kappaB, and AP-1.

Differentially expressed plasma proteins of ß-thalassemia/hemoglobin E patients in response to curcuminoids/vitamin E antioxidant cocktails.

OBJECTIVE: Iron overload and oxidative stress are the major causes of serious complications and mortality in thalassemic patients. Our previous work supports the synergistic effects of antioxidant cocktails (curcuminoids or vitamin E, N-acetylcysteine, and deferiprone) in treatment of ß-thalassemia/Hb E patients. This further 2-DE-based proteomic study aimed to identify the plasma proteins that expressed differentially in response to antioxidant cocktails. METHODS: Frozen plasma samples of ten normal subjects and ten ß-thalassemia/Hb E patients at three-time points (baseline, month 6, and month 12) were reduced the dynamic range of proteome using ProteoMiner kit and separated proteins by two-dimensional gel electrophoresis. Differentially expressed proteins were identified using tandem mass spectrometry. Several plasma proteins were validated by ELISA and Western blot analysis. RESULTS: Thirteen and 11 proteins were identified with altered expression levels in the curcuminoids- and vitamin E cocktail groups, respectively. The associations between vitronectin (VTN) expression and total bilirubin levels, as well as between serum paraoxonase/arylesterase 1 (PON1) expression and blood reactive oxygen species were observed. Validation results were consistent with proteomics results. DISCUSSION AND CONCLUSIONS: These plasma proteins may provide better understanding of the mechanisms underlying the therapeutic effects of antioxidant cocktails in thalassemic patients.

Impact of a group-based treatment program on adipocytokines, oxidative status, inflammatory cytokines and arterial stiffness in obese children and adolescents.

Background Dysregulation of adipocytokines, inflammatory cytokines and oxidative stress are associated with the pathogenesis of obesity-related complications. This study aimed to evaluate the effect of a group-based lifestyle modification program on adipocytokines, inflammatory cytokines, oxidative status and arterial stiffness in obese youth. Methods A 1-year weight-reduction program was conducted. The program consisted of initial hospitalization and five outpatient group-based sessions held at 1, 2, 3, 6 and 9 months. Pre- and post-intervention measurements included anthropometric data, blood tests, body composition and brachial-ankle pulse wave velocity (ba-PWV). Results A total of 126 obese youths were recruited, and 115 of those completed the study. Twenty-four participants had increased percentage weight for height at the end of the study (group A), 30 had minimal reduction (group B) and 61 had substantial reduction (group C). Lean mass significantly increased in all three groups (all p<0.001). A significant decrease in leptin (group A, p=0.021; group B, p=0.005; group C, p<0.001), interleukin-6 (IL-6) (group A, p=0.019; group B, p=0.004; group C, p<0.001) and ba-PWV (group A, p=0.031; group B, p=0.015; group C, p<0.001) was also observed. No significant change in the oxidative status was found among the groups. Reduction in ba-PWV was correlated with decreases in plasma malondialdehyde (pMDA) (r=0.233, p=0.036) and homeostasis model assessment of insulin resistance (HOMA-IR) (r=0.253, p=0.025). Conclusions A group-based healthy lifestyle program for obese youths had beneficial effects on adipocytokines, inflammatory cytokines and arterial stiffness. Participants without change in weight status also benefited. These improvements may reduce the risk of obese youths developing atherosclerosis.

Elevated F2-isoprostanes in thalassemic patients.

This study was aimed at investigating oxidative stress in thalassemic patients by measurement of the oxidative damage biomarker, F(2)-isoprostanes (F(2)-IsoPs), using gas chromatography-mass spectrometry. The results showed that the mean value of urinary F(2)-IsoPs, normalized with creatinine, in the thalassemic group was significantly higher than that from healthy subjects (3.38+/-2.15 ng/mg creatinine vs 0.86+/-0.55 ng/mg creatinine, respectively), and the mean value of plasma total F(2)-IsoPs in the thalassemic group was also significantly higher than that from healthy subjects (0.39+/-0.15 ng/ml vs 0.18+/-0.03 ng/ml, respectively). Serum ferritin, erythrocyte superoxide dismutase (SOD), glutathione peroxidase, glutathione, and TBARS levels after treatment of erythrocytes with H(2)O(2) were also investigated, and serum ferritin and erythrocyte SOD levels were significantly higher in thalassemic patients. Our findings are consistent with oxidative stress in thalassemia patients.

Chicken egg yolk antibodies specific for the gamma chain of human hemoglobin for diagnosis of thalassemia.

Immunoglobulin Y (IgY) technology was used to generate anti-hemoglobin Bart's (Hb Bart's) IgY antibodies (Abs) for development into an enzyme-linked immunosorbent assay (ELISA) test for thalassemia diagnosis. Hb Bart's purified from the hemolysate of a patient with Hb Bart's hydrops fetalis (homozygous alpha-thalassemia) was used to immunize a chicken via the pectoralis muscle. After water dilution and sodium sulfate precipitation, 40 to 70 mg of IgY could be extracted from an egg. IgY, first detected in sera 2 weeks after immunization, reached the highest titer at week 4, and the titer remained stable for at least 2 weeks before declining. The pattern of Ab response in the yolk was the same as in the serum but was somewhat delayed. The IgY Abs produced reacted with gamma globin, Hb Bart's, Hb F, normal cord hemolysate (Hbs F plus A), and Hb Bart's hydrops fetalis (Hbs Bart's plus Portland) and to a lesser degree with beta globin, Hb A, Hb A2 and adult hemolysate (Hbs A plus A2), but the Abs did not react with alpha globin. Immunoaffinity purification with Hb A coupled to Sepharose was used to isolate an unbound IgY that reacted with Hb F, Hb Bart's, and gamma globin, and this IgY was used to develop an ELISA test for thalassemia diagnosis. The results of direct ELISA analyses of 336 hemolysate samples from individuals with various known thalassemia genotypes and phenotypes and from healthy individuals confirmed the specificity of the polyclonal Abs for Hbs containing Hb F and Hb Bart's. This specificity, which was due to the Abs' strong reactivity in cases of pathologic thalassemic diseases and weak reactivity in cases of nonpathologic thalassemic diseases, depended on the levels of Hb Bart's and Hb F.

Camellia Oil-Enriched Diet Attenuates Oxidative Stress and Inflammatory Markers in Hypercholesterolemic Subjects.

Camellia oil is commonly used as an adjuvant in medicine. It is rich in monounsaturated fatty acids, vitamin E, and phytochemicals. The objective of this study was to examine effects of camellia oil consumption on oxidative stress, low-density lipoprotein-cholesterol (LDL-C) oxidation, and inflammatory markers in hypercholesterolemic subjects. The study design was a randomized, single-blind controlled trial. Women with hypercholesterolemia (n = 50) were randomly divided into two groups. The treatment group (n = 25) was provided camellia oil-enriched diets and the control group (n = 25) was provided diets cooked with soybean oil three meals (45 mL oil) a day for 8 weeks. Biomarkers of oxidative stress and inflammatory cytokines were assessed before and the after intervention. Camellia oil consumption significantly decreased malondialdehyde (11.2%; P < .001) whereas glutathione was not changed (P = .382). Moreover, the camellia oil group exhibited a statistically significant decrease in oxidized LDL-C (8.7%; P < .001) compared with the control group. Furthermore, camellia oil consumption significantly decreased high-sensitivity C-reactive protein (12.3%; P < .001) whereas tumor necrosis factor-α and interleukin-6 were not different (P = .079; P = .660, respectively) compared with the control group. These data indicate that the consumption of camellia oil-enriched diet could decrease oxidative stress and inflammatory markers in hypercholesterolemic women. Therefore, camellia oil consumption may reduce cardiovascular disease risk factors.

Effect of coenzyme Q10 as an antioxidant in beta-thalassemia/Hb E patients.

Thalassemia is a group of genetic disorders resulting from different mutations in the globin gene complex and leading to an imbalance in globin synthesis. Unmatched globin chains are less stable and susceptible to oxidation. Patients with beta-thalassemia/HbE are prone to increased oxidative stress as indicated by increased lipid peroxidation product, malondialdehyde (MDA), partly because of the presence of iron in the form of heme and hemichromes released from excess globin chains and excess iron deposition in various tissues. The level of antioxidant such as glutathione is markedly decreased while activities of antioxidant enzymes including superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH-Px) are increased. We have recently found that the levels of coenzyme Q(10) (CoQ(10)) are also very low in thalassemia. We therefore evaluated the oxidative stress and the antioxidants in these patients before and after supplementation with 100 mg CoQ(10) daily for 6 months. The results showed that the plasma level of CoQ(10) significantly increased and the oxidative stress decreased as the level of MDA declined. The administration of CoQ(10) led to significant improvement of biochemical parameters of antioxidant enzymes. The antioxidant supplementation will be beneficial for thalassemia patients as adjunct therapy to increase their quality of life.

Treatment of ß-Thalassemia/Hemoglobin E with Antioxidant Cocktails Results in Decreased Oxidative Stress, Increased Hemoglobin Concentration, and Improvement of the Hypercoagulable State.

Studies on the antioxidant treatment for thalassemia have reported variable outcomes. However, treatment of thalassemia with a combination of hydrophobic and hydrophilic antioxidants and an iron chelator has not been studied. This study investigated the effects of antioxidant cocktails for the treatment of ß-thalassemia/hemoglobin E (HbE), which is the most common form of ß-thalassemia in Southeast Asia. Sixty patients were divided into two groups receiving N-acetylcysteine, deferiprone, and either curcuminoids (CUR) or vitamin E (Vit-E), and their hematological parameters, iron load, oxidative stress, and blood coagulation potential were evaluated. Patients were classified as responders if they showed the improvements of the markers of iron load and oxidative stress, otherwise as nonresponders. During treatment, the responders in both groups had significantly decreased iron load, oxidative stress, and coagulation potential and significantly increased antioxidant capacity and hemoglobin concentration. The significantly maximum increase (P < 0.01) in hemoglobin concentration was 11% at month 4 in CUR group responders and 10% at month 10 in Vit-E group responders. In conclusion, the two antioxidant cocktails can improve anemia, iron overload, oxidative stress, and hypercoagulable state in ß-thalassemia/HbE.

Role of curcuminoids in ameliorating oxidative modification in ß-thalassemia/Hb E plasma proteome.

Thalassemic patients often exhibit high levels of oxidative stress and iron overload, which can lead to hazardous complications. Curcuminoids, extracted from the spice turmeric, are known to have antioxidant and iron-chelating properties and have been proposed as a potential upstream therapy of thalassemia. Here we have applied proteomic techniques to study the protein profile and oxidative damage in the plasma of ß-thalassemia/Hb E patients before and after treatment with curcuminoids. In this study, 10 ß-thalassemia/Hb E patients were treated with 500 mg curcuminoids daily for 12 months. The plasma protein profile and protein carbonyl content were determined at baseline, 6 and 12 months using two-dimensional fluorescence difference gel electrophoresis and carbonyl immunoblotting, respectively. Other hematological, clinical, and biochemical parameters were also analyzed. Twenty-six spots, identified as coagulation factors and proteins involved in iron homeostasis, showed significantly decreased intensity in thalassemic plasma, compared to those of normal subjects. Treatment with curcuminoids up-regulated the plasma levels of these proteins and reduced their oxidative damage. Serum non-transferrin bound iron, platelet factor-3 like activity, oxidative stress parameters and antioxidant enzymes were also improved after curcuminoids treatment. This study is the first proteomic study of plasma in the thalassemic state and also shows the ameliorating role of curcuminoids towards oxidative stress and iron overload in the plasma proteome.

Improvement in oxidative stress and antioxidant parameters in beta-thalassemia/Hb E patients treated with curcuminoids.

OBJECTIVES: To evaluate the hematological profile, oxidative stress, and antioxidant parameters in beta-thalassemia/Hb E patients treated with curcuminoids for 12 months. DESIGN AND METHODS: Twenty-one beta-thalassemia/Hb E patients were given 2 capsules of 250 mg each of curcuminoids (a total of 500 mg) daily for 12 months. Blood was collected every 2 months during treatment and 3 months after withdrawal and was determined for complete blood count, malonyldialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), reduced glutathione (GSH) in red blood cells (RBC), and non-transferrin bound iron (NTBI) in serum. RESULTS: The increased oxidative stress in beta-thalassemia/Hb E patients was shown by higher levels of MDA, SOD, GSH-Px in RBC, serum NTBI, and lower level of RBC GSH. Curcuminoids administration resulted in improvement of all the measured parameters as long as they were administered. After 3 months withdrawal of treatment, all parameters returned close to baseline levels. CONCLUSION: Curcuminoids may be used to ameliorate oxidative damage in patients with beta-thalassemia/Hb E disease.

Bioaccessibility, biotransformation, and transport of alpha-mangostin from Garcinia mangostana (Mangosteen) using simulated digestion and Caco-2 human intestinal cells.

alpha- and gamma-Mangostin are the most abundant prenylated xanthones present in the fruit of the mangosteen tree. These compounds have been reported to possess numerous bioactivities that have provided the impetus for use of mangosteen products as nutraceuticals and in functional foods and dietary supplements. The health-promoting benefits of mangosteen are dependent on delivery of the xanthones to target tissues. Here, we used simulated digestion and Caco-2 cells to investigate the digestive stability, bioaccessibility, and intestinal cell transport of alpha- and gamma- mangostin. Recovery of alpha- and gamma-mangostin after simulated digestion of pericarp and fruit pulp exceeded 90%. Transfer of alpha- and gamma-mangostin to the aqueous fraction during simulated digestion was efficient (65-74%) and dependent on bile salts suggesting that micellarization is required for optimal bioaccessibility of xanthones. Cell uptake of xanthones from micelles was dose dependent and intracellular concentrations were maximum by 1 h. Both free and phase II metabolites of alpha-mangostin were transported in the basolateral compartment and metabolites also effluxed into the apical chamber. Transepithelial transport of alpha-mangostin was increased during prandial-like compared to fasted conditions suggesting that absorption is enhanced by dietary fat.

Quantitative determination of ortho- and meta-tyrosine as biomarkers of protein oxidative damage in beta-thalassemia.

Oxidative stress in thalassemia is caused by secondary iron overload and stems from blood transfusion and increased iron uptake. In this study, we hypothesized that levels of o- and m-tyrosine, products of hydroxyl radical attack on phenylalanine, would be elevated in beta-thalassemia (intermediate). This study represents the first report in which specific markers of protein oxidative damage have been quantified in thalassemia. We used GC/MS to assay o- and m-tyrosine at the femtomole level using only a few microliters of plasma. Levels of both markers were significantly higher in patients with beta-thalassemia than in controls and were positively correlated with serum ferritin, malondialdehyde, superoxide dismutase, glutathione peroxidase and glutathione. We conclude that o- and m-tyrosine are useful biomarkers of oxidative damage to proteins in thalassemia (intermediate) and may also be useful markers in other iron overload diseases. Positive correlations between o- and m-tyrosine levels and malondialdehyde as well as antioxidants such as superoxide dismutase, glutathione peroxidase and glutathione, are indicative of the broad impact of oxidative stress on blood plasma in thalassemia, with up-regulation of antioxidant proteins probably reflecting a homeostatic response to these increased stress levels.

Chicken Egg Yolk Antibodies Specific for the γ Chain of Human Hemoglobin for Diagnosis of Thalassemia.

Immunoglobulin Y (IgY) technology was used to generate anti-hemoglobin Bart's (Hb Bart's) IgY antibodies (Abs) for development into an enzyme-linked immunosorbent assay (ELISA) test for thalassemia diagnosis. Hb Bart's purified from the hemolysate of a patient with Hb Bart's hydrops fetalis (homozygous α-thalassemia) was used to immunize a chicken via the pectoralis muscle. After water dilution and sodium sulfate precipitation, 40 to 70 mg of IgY could be extracted from an egg. IgY, first detected in sera 2 weeks after immunization, reached the highest titer at week 4, and the titer remained stable for at least 2 weeks before declining. The pattern of Ab response in the yolk was the same as in the serum but was somewhat delayed. The IgY Abs produced reacted with γ globin, Hb Bart's, Hb F, normal cord hemolysate (Hbs F plus A), and Hb Bart's hydrops fetalis (Hbs Bart's plus Portland) and to a lesser degree with ß globin, Hb A, Hb A2, and adult hemolysate (Hbs A plus A2), but the Abs did not react with α globin. Immunoaffinity purification with Hb A coupled to Sepharose was used to isolate an unbound IgY that reacted with Hb F, Hb Bart's, and γ globin, and this IgY was used to develop an ELISA test for thalassemia diagnosis. The results of direct ELISA analyses of 336 hemolysate samples from individuals with various known thalassemia genotypes and phenotypes and from healthy individuals confirmed the specificity of the polyclonal Abs for Hbs containing Hb F and Hb Bart's. This specificity, which was due to the Abs' strong reactivity in cases of pathologic thalassemic diseases and weak reactivity in cases of nonpathologic thalassemic diseases, depended on the levels of Hb Bart's and Hb F.