Supplementation with red palm oil increases ß-carotene and vitamin A blood levels in patients with cystic fibrosis.

Patients with cystic fibrosis (CF) show decreased plasma concentrations of antioxidants due to malabsorption of lipid soluble vitamins and consumption by chronic pulmonary inflammation. ß-Carotene is a major source of retinol and therefore is of particular significance in CF. The aim of this study was to investigate the effect of daily intake of red palm oil (RPO) containing high amounts of ß-carotene on the antioxidant levels in CF patients. Sixteen subjects were recruited and instructed to enrich their food with 2 to 3 tablespoons of RPO (~1.5 mg of ß-carotene) daily over 8 weeks. Carotenoids, retinol, and α-tocopherol were measured in plasma at baseline and after intervention. In addition ß-carotene, lycopene, α-tocopherol, and vitamin C were measured in buccal mucosa cells (BMC) to determine the influence of RPO on antioxidant tissue levels. Eleven subjects completed the study properly. Plasma ß-carotene, retinol, and α-carotene of these patients increased, but plasma concentrations of other carotenoids and α-tocopherol as well as concentrations of ß-carotene, lycopene, α-tocopherol, and vitamin C in BMC remained unchanged. Since RPO on a daily basis did not show negative side effects the data suggest that RPO may be used to elevate plasma ß-carotene in CF.

Peter Eckl: Research on the Pro-/Antioxidant Balance.

Peter Maria ECKL started his scientific career in the late 1970s at the Paris-Lodron University of Salzburg working in the field of radiation research [...].

CFTR Modulator Therapy with Lumacaftor/Ivacaftor Alters Plasma Concentrations of Lipid-Soluble Vitamins A and E in Patients with Cystic Fibrosis.

RATIONALE: Cystic fibrosis (CF), caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, leads to impaired pancreatic function and therefore reduced intestinal absorption of lipids and fat-soluble vitamins especially in patients with CF developing pancreatic insufficiency (PI). Previous studies showed that CFTR modulator therapy with lumacaftor-ivacaftor (LUM/IVA) in Phe508del-homozygous patients with CF results in improvement of pulmonary disease and thriving. However, the effects of LUM/IVA on plasma concentration of the lipid soluble vitamins A and E remain unknown. OBJECTIVES: To investigate the course of plasma vitamin A and E in patients with CF under LUM/IVA therapy. METHODS: Data from annual follow-up examinations of patients with CF were obtained to assess clinical outcomes including pulmonary function status, body mass index (BMI), and clinical chemistry as well as fat-soluble vitamins in Phe508del-homozygous CF patients before initiation and during LUM/IVA therapy. RESULTS: Patients with CF receiving LUM/IVA improved substantially, including improvement in pulmonary inflammation, associated with a decrease in blood immunoglobulin G (IgG) from 9.4 to 8.2 g/L after two years (p < 0.001). During the same time, plasma vitamin A increased significantly from 1.2 to 1.6 µmol/L (p < 0.05), however, levels above the upper limit of normal were not detected in any of the patients. In contrast, plasma vitamin E as vitamin E/cholesterol ratio decreased moderately over the same time from 6.2 to 5.5 µmol/L (p < 0.01). CONCLUSIONS: CFTR modulator therapy with LUM/IVA alters concentrations of vitamins A and vitamin E in plasma. The increase of vitamin A must be monitored critically to avoid hypervitaminosis A in patients with CF.

Metabolism of 4-hydroxy-2-nonenal in human polymorphonuclear leukocytes.

Intracellular metabolism of 4-hydroxy-2-nonenal (HNE), a major product and mediator of oxidative stress and inflammation, is analyzed in resting and fMLP-stimulated human polymorphonuclear leukocytes (PMNL), where this compound is generated during activation of the respiratory burst. HNE consumption rate in PMNL is very low, if compared to other cell types (rat hepatocytes, rabbit fibroblasts), where HNE metabolism is always an important part of secondary antioxidative defense mechanisms. More than 98% of HNE metabolites are identified. The pattern of HNE intermediates is quite similar in stimulated and resting PMNL - except for higher water formation in resting PMNL - while the initial velocity of HNE degradation is somewhat higher in resting cells, 0.44 instead of 0.28 nmol/(min×10(6) cells). The main products of HNE metabolism are 4-hydroxynonenoic acid (HNA), 1,4-dihydroxynonene (DHN) and the glutathione adducts with HNE, HNA, and DHN. Protein-bound HNE and water account for about 3-4% of the total HNE derivatives in stimulated cells, while in resting cells protein-bound HNE and water are 4% and 20%, respectively. Cysteinyl-glycine-HNE adduct and mercapturic acids contribute to about 5%.

In Vitro Aging of Human Skin Fibroblasts: Age-Dependent Changes in 4-Hydroxynonenal Metabolism.

Evidence suggests that the increased production of free radicals and reactive oxygen species lead to cellular aging. One of the consequences is lipid peroxidation generating reactive aldehydic products, such as 4-hydroxynonenal (HNE) that modify proteins and form adducts with DNA bases. To prevent damage by HNE, it is metabolized. The primary metabolic products are the glutathione conjugate (GSH-HNE), the corresponding 4-hydroxynonenoic acid (HNA), and the alcohol 1,4-dihydroxynonene (DHN). Since HNE metabolism can potentially change during in vitro aging, cell cultures of primary human dermal fibroblasts from several donors were cultured until senescence. After different time points up to 30 min of incubation with 5 µM HNE, the extracellular medium was analyzed for metabolites via liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI-MS). The metabolites appeared in the extracellular medium 5 min after incubation followed by a time-dependent increase. But, the formation of GSH-HNL and GSH-DHN decreased with increasing in vitro age. As a consequence, the HNE levels in the cells increase and there is more protein modification observed. Furthermore, after 3 h of incubation with 5 µM HNE, younger cells showed less proliferative capacity, while in older cells slight increase in the mitotic index was noticed.

Improvement in skin elasticity in the treatment of cellulite and connective tissue weakness by means of extracorporeal pulse activation therapy.

BACKGROUND: Extracorporeal pulse activation therapy (EPAT), also called extracorporeal acoustic wave therapy, seeks to achieve effective and long-lasting improvement of age-related connective tissue weakness in the extremities, especially in the treatment of unsightly cosmetic skin defects referred to as cellulite. OBJECTIVE: The objective of this study was to stimulate metabolic activity in subcutaneous fat tissue by means of EPAT in order evaluate its effectiveness in enhancing connective tissue firmness and improving skin texture and structure. METHODS: Fifty-nine women with advanced cellulite were divided into 2 groups; one group of 15 patients received planar acoustic wave treatment for 6 therapy sessions within 3 weeks; a second group of 44 patients received 8 therapy sessions within 4 weeks. Changes in connective tissue were evaluated using the DermaScan C ultrasound system (Cortex Technology, Hadsund, Denmark). Skin elasticity measurements were performed using the DermaLab system (Cortex Technology). Photographs of treated areas were taken at each therapy session and at follow-up sessions. RESULTS: Skin elasticity values gradually improved over the course of EPAT therapy and revealed a 73% increase at the end of therapy. At 3- and 6-month follow-ups, skin elasticity had even improved by 95% and 105%, respectively. Side effects included minor pain for 3 patients during therapy and slight skin reddening. CONCLUSIONS: This study confirmed the effects of acoustic wave therapy on biologic tissue, including stimulation of microcirculation and improvement of cell permeability. Ultrasound evaluation demonstrated increased density and firmness in the network of collagen/elastic fibers in the dermis and subcutis. Treatment was most effective in older patients with a long history of cellulite.

4-Hydroxynonenal (HNE) modified proteins in metabolic diseases.

4-Hydroxynonenal (HNE) is one of the quantitatively most important products of lipid peroxidation. Due to its high toxicity it is quickly metabolized, however, a small share of HNE avoids enzymatic detoxification and reacts with biomolecules including proteins. The formation of HNE-protein-adducts is one of the accompanying processes in oxidative stress or redox disbalance. The modification of proteins might occur at several amino acids side chains, leading to a variety of products and having effects on the protein function and fate. This review summarizes current knowledge on the formation of HNE-modified proteins, their fate in mammalian cells and their potential role as a damaging agents during oxidative stress. Furthermore, the potential of HNE-modified proteins as biomarkers for several diseases are highlighted.

Clinical oxidation parameters of aging.

Aging is a complex progressive physiological alteration of the organism which ultimately leads to death. During the whole life a human being is confronted with oxidative stress. To measure how this oxidative stress is developing during the aging process and how it changes the cellular metabolism several substances have been pronounced as biomarkers including lipid peroxidation (LPO) products, protein oxidation products, antioxidative acting enzymes, minerals, vitamins, glutathione, flavonoids, bilirubin and uric acid (UA). But none of them could develop to the leading one which is accepted by the whole scientific community to determine the life expectancy of the individual person or biological age or age-related health status. Further there are many conflicting data about the changes of each single biomarker during the aging process. There are so many different influences acting on the concentration or activity of single substances or single enzymes that it is not possible to measure only one clinical marker and determine how healthy an individual is or to predict the life expectancy of the corresponding person. Therefore, always a set or pattern of clinical biomarkers should be used to determine the oxidation status of the person. This set should include at least one marker for the LPO, the protein oxidation and the total antioxidative status and ideally also one for DNA damages.

Age-associated analysis of oxidative stress parameters in human plasma and erythrocytes.

Oxidative damage accumulation in macromolecules has been considered as a cause of cellular damage and pathology. Rarely, the oxidative stress parameters in healthy humans related to the individual age have been reported. The purpose of this study was to examine the redox status in plasma and erythrocytes of healthy individuals and determine correlations between these parameters and the aging process. The following parameters were used: malondialdehyde (MDA), protein carbonyls (PCO), 4-hydroxy-2,3-trans-nonenal (HNE), reduced glutathione (GSH), glutathione disulfide (GSSG) and uric acid (UA) in blood and plasma samples of 194 healthy women and men of ages ranging from 18 to 84 years. The results indicate that the balance of oxidant and antioxidant systems in plasma shifts in favor of accelerated oxidation during ageing. That is demonstrated by increases of MDA, HNE, GSSG and by the slight decrease of erythrocytic GSH with age. As the content of UA is more determined by metabolic and nutritional influences than by the balance between prooxidants and antioxidants there was no significant age-related change observed. For plasma concentrations of HNE the first time age-dependent reference values for healthy humans are presented.

Carotenoid cleavage products modify respiratory burst and induce apoptosis of human neutrophils.

Carotenoid supplementation in the treatment of diseases associated with oxidative stress has been recently questioned because of the cell damage and the increased risk of lung cancer in male smokers. Because of the complex role of neutrophils in lung diseases, we investigated whether carotenoid derivatives could affect respiratory burst and apoptosis of human neutrophils purified from peripheral blood. Stimulation of superoxide production was induced by nanomolar and micromolar concentrations of carotenoid cleavage products with aliphatic chains of different length, but not by carotenoids lacking the carbonyl moiety. The stimulatory effect of carotenoid cleavage products was observed in cells activated by phorbol myristate acetate (PMA), while a slight inhibition of superoxide production was noticed with cells activated by the chemotactic tripeptide N-formyl-Met-Leu-Phe (f-MLP). At higher concentrations, carotenoid cleavage products inhibited superoxide production in the presence of both PMA and f-MLP. In the presence of 20 microM carotenoid cleavage products, inhibition of superoxide production was accompanied by DNA fragmentation and increased level of intracellular caspase-3 activity.

Beta-carotene cleavage products induce oxidative stress in vitro by impairing mitochondrial respiration.

Carotenoids are widely used as important micronutrients in food. Furthermore, carotenoid supplementation has been used in the treatment of diseases associated with oxidative stress. However, in some clinical studies harmful effects have been observed, for example, a higher incidence of lung cancer in individuals exposed to extraordinary oxidative stress. The causal mechanisms are still unclear. Carotenoid cleavage products (CCPs), including highly reactive aldehydes and epoxides, are formed during oxidative attacks in the course of antioxidative action. Here, we tested the hypothesis that CCPs may increase oxidative stress by impairing mitochondrial function. We found that CCPs strongly inhibit state 3 respiration of isolated rat liver mitochondria even at concentrations between 0.5 and 20 microM. This was true for retinal, beta-ionone, and mixtures of cleavage products, which were generated in the presence of hypochlorite to mimic their formation in inflammatory regions. The inhibition of mitochondrial respiration was accompanied by a reduction in protein sulfhydryl content, decreasing glutathione levels and redox state, and elevated accumulation of malondialdehyde. Changes in mitochondrial membrane potential favor functional deterioration of the adenine nucleotide translocator. The findings may reflect a basic mechanism of increasing the risk of cancer induced by CCPs.

Beta-carotene breakdown products may impair mitochondrial functions--potential side effects of high-dose beta-carotene supplementation.

Beta-carotene (BC) and other carotenoids are mainly considered as belonging to the group of micronutrients. As they are contained in fruit and vegetables and thus part of human diet, a regular low-dose intake from natural sources is normally assured. In the last decade high-dose supplementation with synthetic carotenoids has been used successfully in the treatment of diseases believed to be associated with oxidative stress. However, in a few clinical studies harmful effects have been observed as well, e.g., a higher incidence of lung cancer after BC was given in high doses to smokers. Our studies aim at shedding light on the causal mechanisms of the known side effects that we have investigated. Possibilities of preventing them are discussed. Obviously, on certain conditions of high-dose carotenoid supplementation, both the antioxidant and prooxidant reactions may arise. Carotenoid breakdown products (CBP) including very reactive aldehydes and epoxides are formed during oxidative attack in the course of antioxidative action. Carotenoid breakdown products inhibit state 3 respiration of isolated rat liver mitochondria at concentrations between 0.5 and 20 microM. In vivo stimulated neutrophils might represent an important source for the generation of CBP, and the lung might be a critical organ in CBP formation. The inhibition of mitochondrial state 3 respiration by CBP is accompanied by a reduced content of protein sulfhydryl groups, decreasing glutathione levels and redox state, and also elevated accumulation of malondialdehyde. Changes in mitochondrial membrane potential favour functional deterioration of the adenine nucleotide translocator (ANT). The findings reflect a basic mechanism of the side effects of BC supplementation in circumstances of severe oxidative stress induced by CBP representing a class of lipid oxidation products. We are striving for safe conditions of carotenoid supplementation in order to protect patients in need of this kind of medical treatment from possible side effects, such as unwanted prooxidative reactions.

Anti-fibrosclerotic effects of shock wave therapy in lipedema and cellulite.

In vivo measurements in 26 female patients with lipedema and cellulite parameters were carried out before and after therapy by means of complex physical decongestive therapy (CPDT) including manual lymph drainage and compression as main components and/or shock wave therapy (SWT). Oxidative stress parameters of blood serum and biomechanic skin properties/smoothening of dermis and hypodermis surface were evaluated. Oxidative stress in lipedema and cellulite was demonstrated by increased serum concentrations of malondialdehyde (MDA) and plasma protein carbonyls compared with healthy control persons. Both MDA and protein carbonyls in blood plasma decreased after serial shock wave application and CPDT. The SWT itself and CPDT itself lead to MDA release from edematous tissue into the plasma. Obviously both therapy types, SWT and CPDT, mitigate oxidative stress in lipedema and cellulite. In parallel SWT improved significantly the biomechanic skin properties leading to smoothening of dermis and hypodermis surface. Significant correlation between MDA depletion of edematous and lipid enriched dermis and improvement of mechanic skin properties was demonstrated. From these findings it is concluded, that a release of lipid peroxidation (LPO) products from edematous dermis is an important sclerosis-preventing effect of SWT and/or CPDT in lipedema and cellulite. Expression of factors stimulating angiogenesis and lymphangiogenesis such as VEGF was not induced by SWT and/or CPDT and, therefore, not involved in beneficial effects by SWT and/or CPDT.

F2-isoprostanes as biomarkers of lipid peroxidation in patients with chronic renal failure.

Chronic renal failure patients on long-term hemolysis are found to be under increased oxidative stress, caused by antioxidant deficiency, neutrophil activation during hemodialysis (HD), platelet activation and/or chronic inflammation. Increased levels of oxidants (e.g. malondialdehyde, 4-hydroxynonenal, hydrocarbons, lipohydroperoxides, oxycholesterols, carbonyls) in HD patients are thought to play an important role in the development of endothelial dysfunction, atherogenesis and cardiovascular disease, which is a frequent condition in end-stage renal disease. F2-isoprostanes have been established as chemically stable, highly specific and reliable biomarkers of in vivo oxidative stress which can very sensitively measured by gas chromatography-mass spectrometry (Morrow et al. [17]). An up to 6-fold increase of plasma F2-isoprostanes in HD patients is accompanied by an enhanced formation of indicators of inflammation (e.g. C-reactive protein) and decreases of endogenous antioxidants (e.g. ascorbate, alpha-tocopherol). In their esterified form F2-isoprostanes may be a useful criteria to evaluate the effectiveness of clinical interventions to diminish oxidant stress and associated inflammation. Furthermore, F2-isoprostanes possess potent biological activities (e.g. 8-iso-PGF2alpha is known as a renal vasoconstrictor) suggesting that they may also act as mediators of the cellular effects of oxidative stress and inflammation.

4-Hydroxy-nonenal-A Bioactive Lipid Peroxidation Product.

This review on recent research advances of the lipid peroxidation product 4-hydroxy-nonenal (HNE) has four major topics: I. the formation of HNE in various organs and tissues, II. the diverse biochemical reactions with Michael adduct formation as the most prominent one, III. the endogenous targets of HNE, primarily peptides and proteins (here the mechanisms of covalent adduct formation are described and the (patho-) physiological consequences discussed), and IV. the metabolism of HNE leading to a great number of degradation products, some of which are excreted in urine and may serve as non-invasive biomarkers of oxidative stress.

Intracellular metabolism of 4-hydroxynonenal.

4-hydroxynonenal (HNE) is a major aldehydic product of lipid peroxidation known to exert a multitude of biological, cytotoxic, and signal effects. Mammalian cells possess highly active pathways of HNE metabolism. The metabolic fate of HNE was investigated in various mammalian cells and organs such as hepatocytes, intestinal enterocytes, renal tubular cells, aortic and brain endothelial cells, synovial fibroblasts, neutrophils, thymocytes, heart, and tumor cells. The experiments were carried out at 37 degrees C at initial HNE concentrations between 1 microM--that means in the range of physiological and pathophysiologically relevant HNE levels--to 100 microM. In all cell types which were investigated, 90-95% of 100 microM HNE were degraded within 3 min of incubation. At 1 microM HNE the physiological blood serum level of about 0.1-0.2 microM was restored already after 10-30 s. As primary products of HNE in hepatocytes and other cell types the glutathione-HNE-1:1-conjugate, the hydroxynonenoic acid and the corresponding alcohol of HNE, the 1,4-dihydroxynonene, were identified. Furthermore, the beta-oxidation of hydroxynonenoic acid including the formation of water was demonstrated. The quantitative share of HNE binding to proteins was low with about 2-8% of total HNE consumption. The glycine-cysteine-HNE, cysteine-HNE adducts and the mercapturic acid from glutathione-HNE adduct were not formed in the most cell types, but in kidney cells and neutrophils. The rapid metabolism underlines the role of HNE degrading pathways in mammalian cells as important part of the secondary antioxidative defense mechanisms in order to protect proteins from modification by aldehydic lipid peroxidation products.

Beta-carotene cleavage products after oxidation mediated by hypochlorous acid--a model for neutrophil-derived degradation.

After beta-carotene failed in certain clinical efficacy trials, there is evidence that the carotenoid might even be harmful, especially to smokers, when given in high dosages. These negative effects might be mediated in part also by carotenoid cleavage products (CPs) having a high reactivity towards biomolecules. The authors postulate that in certain tissues oxidative, nonenzymatic cleavage of carotenoids is carried out primarily by oxidants liberated by polymorphonuclear leukocytes (PML). In this study, we show that beta-carotene is degraded by stimulated PML in vitro. This gives the pathophysiological meaning to our further experiments in which beta-carotene degradation by hypochlorous acid and consecutive CP formation were investigated. While formation of apo-carotenals under these conditions has been studied before, this was not the case for short chain products. Performing gas chromatography mass spectrometry, we were able to identify for the first time 5,6-epoxi-beta-ionone, ionene, beta-cyclocitral, beta-ionone, dihydroactinidiolide, and 4-oxo-beta-ionone as CPs formed after degradation of beta-carotene mediated by hypochlorous acid. Our findings may be of biological relevance because beta-carotene CPs are highly reactive and, therefore, potentially toxic.

Oxidation of carotenoids by heat and tobacco smoke.

The stability to autoxidation of the polar carotenoids, lutein and zeaxanthin, was compared to that of the less polar carotenoids, beta-carotene and lycopene at physiologically or pathophysiologically relevant concentrations of 2 and 6 microM, after exposure to heat or cigarette smoke. Three methodological approaches were used: 1) Carotenoids dissolved in solvents with different polarities were incubated at 37 and 80 degrees C for different times. 2) Human plasma samples were subjected to the same temperature conditions. 3) Methanolic carotenoid solutions and plasma were also exposed to whole tobacco smoke from 1-5 unfiltered cigarettes. The concentrations of individual carotenoids in different solvents were determined spectrophotometrically. Carotenoids from plasma were extracted and analyzed using high performance liquid chromatography. Carotenoids were generally more stable at 37 than at 80 degrees C. In methanol and dichloromethane the thermal degradation of beta-carotene and lycopene was faster than that of lutein and zeaxanthin. However, in tetrahydrofuran beta-carotene and zeaxanthin degraded faster than lycopene and lutein. Plasma carotenoid levels at 37 degrees C did not change, but decreased at 80 degrees C. The decrease of beta-carotene and lycopene levels was higher than those for lutein and zeaxanthin. Also in the tobacco smoke experiments the highest autoxidation rates were found for beta-carotene and lycopene at 2 microM, but at 6 microM lutein and zeaxanthin depleted to the same extent as beta-carotene. These data support our previous studies suggesting that oxidative stress degrade beta-carotene and lycopene faster than lutein and zeaxanthin. The only exception was the thermal degradation of carotenoids solubilized in tetrahydrofuran, which favors faster breakdown of beta-carotene and zeaxanthin.

Oxidative stress and antioxidants in carcinogenesis and integrative therapy of cancer.

Oxidative stress is often considered as a causative factor in carcinogenesis. In addition, current knowledge recognizes oxidative stress as a mechanism by which various cancer therapies act against cancer. To ameliorate the side effects of cancer therapy, many of the patients suffering from cancer are subject to adjuvant therapy, which often implies antioxidant supplementation. Yet, the benefits of such adjuvant treatments are still uncertain owing to the lack of appropriate integrative and personalized medical approach. In particular, reactive oxygen species formed during oxidative stress and products of lipid peroxidation are not only cytotoxic, but can modulate signal transduction in cells, which also behave similar to individuals under stress. Accordingly, pro-oxidants and antioxidants might be considered as modifiers of specific cellular redox signaling. Therefore, there is a need to evaluate the potential benefits of antioxidant supplements in healthy persons, and in particular in cancer patients during therapy. Our review will present a summary of the existing knowledge regarding the effects of various antioxidants in cancer therapies, focusing on cellular adaptation to oxidative stress interacting with redox signaling transduction pathways thereby influencing cell growth.