Anna Maria Pintaudi

Melatonin protects human red blood cells from oxidative hemolysis: new insights into the radical-scavenging activity.

Abstract: Antioxidant activity of melatonin in human erythrocytes, exposed to oxidative stress by cumene hydroperoxide (cumOOH), was investigated. CumOOH at 300 μM progressively oxidized a 1% suspension of red blood cells (RBCs), leading to 100% hemolysis in 180 min. Malondialdehyde and protein carbonyls in the membrane showed a progressive increase, as a result of the oxidative damage to membrane lipids and proteins, reaching peak values after 30 and 40 min, respectively. The membrane antioxidant vitamin E and the cytosolic reduced glutathione (GSH) were totally depleted in 20 min. As a consequence of the irreversible oxidative damage to hemoglobin (Hb), hemin accumulated into the RBC membrane during 40 min. Sodium dodecyl sulfate (SDS) gel electrophoresis of membrane proteins showed a progressive loss of the cytoskeleton proteins and formation of low molecular weight bands and protein aggregates, with an increment of the intensity of the Hb band. Melatonin at 50 μM strongly enhanced the RBC resistance to oxidative lysis, leading to a 100% hemolysis in 330 min. Melatonin had no effect on the membrane lipid peroxidation, nor prevented the consumption of glutathione (GSH) or vitamin E. However, it completely inhibited the formation of membrane protein carbonyls for 20 min and hemin precipitation for 10 min. The electrophoretic pattern provided further evidence that melatonin delayed modifications to the membrane proteins and to Hb. In addition, RBCs incubated for 15 min with 300 μM cumOOH in the presence of 50 μM melatonin were less susceptible, when submitted to osmotic lysis, than cells incubated in its absence. Extraction and high‐performance liquid chromatography (HPLC) analysis showed a much more rapid consumption of melatonin during the first 10 min of incubation, then melatonin slowly decreased up to 30 min and remained stable thereafter. Equilibrium partition experiments showed that 15% of the melatonin in the incubation mixture was recovered in the RBC cytosol, and no melatonin was extracted from RBC membrane. However, 35% of the added melatonin was consumed during RBC oxidation. Hydroxyl radical trapping agents, such as dimethylsulfoxide or mannitol, added into the assay in a 1,000 times molar excess, did not vary melatonin consumption, suggesting that hydroxyl radicals were not involved in the indole consumption. Our results indicate that melatonin is actively taken up into crythrocytes under oxidative stress, and is consumed in the defence of the cell, delaying Hb denaturation and release of hemin. RBCs are highly exposed to oxygen and can be a site for radical formation, under pathological conditions, which results in their destruction. A protective role of melatonin should be explored in hemolytic diseases.

Contribution of vitamin A to the oxidation resistance of human low density lipoproteins

This study investigated the antioxidant contribution of vitamin A in protecting human low density lipoprotein (LDL) against copper-stimulated oxidation. The presence of small amounts of retinol (0.033 +/- 0.012 nmol/mol LDL) and retinyl palmitate (0.036 +/- 0.021 nmol/mol LDL) was routinely ascertained in the LDL. A single oral supplementation with 20,000 IU vitamin A caused a two- to three-fold increase of retinol and retinyl palmitate in the LDL isolated 8 h after the supplementation. In comparison to autologous-control LDL, vitamin A-enriched LDL were more resistant to oxidation, as expressed both by a clear delay in the onset of lipid peroxidation and by a reduction of the rate of conjugated diene hydroperoxide production during the propagation phase. The calculated incremental increase in the lag phase produced by 1 mol retinol per mol LDL is about 1000 min, suggesting that retinol is more potent than alpha-tocopherol in LDL. Oxidation experiments carried out with LDL isolated from plasma incubated in vitro with either retinol or retinyl palmitate indicated that retinol does lengthen the lag phase, whereas retinyl palmitate can slow the rate of peroxyl chain propagation, without affecting the duration of the lag phase. Temporal disappearance of retinol and retinyl palmitate, followed in comparison with that of alpha-tocopherol and beta-carotene, indicated that the reactivity of the antioxidants with lipoperoxyl radicals was in the sequence alpha-tocopherol, retinol, beta-carotene, and retinyl esters. Although the detailed antioxidant mechanism remains to be elucidated, these results suggest that LDL-associated vitamin A can play a role in maintaining the antioxidant status of LDL during oxidative stress in vivo.

Oxidative stress after moderate to extensive burning in humans

Lipid peroxidation products, lipid antioxidants, and hematologic and blood chemistry changes were evaluated in plasma of patients after acute burning injury involving 10% (n=8), 20% (n=8), and 40% (n=5) of total body surface area (TBSA), 24 h after burning (baseline) up to 30 days after. Markedly increased plasma levels of malondialdehyde (MDA) were observed at baseline in all patients, according to the extent of the injury, then the values declined progressively. However, levels of MDA remained above normal up to 30 days even in less injured patients. On the other hand, the plasma level of conjugated diene lipid hydroperoxides was only slightly higher than control at the baseline, then dropped under the control value in all patients. Cholesterol showed a marked fall at baseline, followed by a rapidly progressive decrease, indicating a massive loss of circulating lipids by the acute thermal injury. Because of such an extensive and rapidly spreading oxidative degradation of lipids, decomposition of conjugated diene hydroperoxides, produced in early stages of the peroxidation process, occurs, so these compounds cannot be a suitable index to value lipid oxidation in burned patients. Aldehydic products of lipid peroxidation act as endotoxins, causing damage to various tissues and organs. Damage to liver and decrease of erythrocyte survival were assessed by increased plasma levels of asparate and alanine transaminases, within 7–15 days after injury, and by a decreased number of red blood cells, which remained under the normal value at 30 days. A marked decrease of lipid antioxidants, β-carotene, vitamin A and vitamin E was observed at baseline. The level of β-carotene remained low in all patients at the end of the 30-day observation. A complete recovery of vitamin A did not occur at 30 days post-burn, even in the patients with 10% of burned TBSA. Plasma levels of vitamin E decreased significantly in 1–7 days after burn in all patients, but these levels increased thereafter, with almost total recovery at 30 days. These data show evidence of a marked, long-lasting oxidant/antioxidant imbalance in burned patients, in accordance with the severity of the injury, which is also reflected as systemic oxidant stress.

Exposure to malondialdehyde induces an early redox unbalance preceding membrane toxicity in human erythrocytes.

This work investigated the oxidative injury to human red blood cells (RBCs) by the exposure to exogenous malondialdehyde (MDA), in a physiological environment. When a 10% RBC suspension was incubated in autologous plasma, in the presence of 50 u w M MDA, 30% of MDA entered into the cells. A time-course study showed that MDA caused early (30-120 u min) and delayed (3-18 u h) effects. MDA caused a fast depletion of reduced glutathione, and loss of the glucose-6-phosphate dehydrogenase activity, followed by a decrease of HbO 2 . Accumulation of methemoglobin, and formation of small amounts of hemichrome were later evident. Also, an HbO 2 -derived fluorescent product was measured in the membrane. The redox unbalance was followed by structural and functional damage to the membrane, evident as the formation of conjugated diene lipid hydroperoxides, concurrent with a sharp accumulation of MDA, consumption of membrane vitamin E, and egress of K + ions. SDS--PAGE of membrane proteins showed formation of high molecular weight aggregates. In spite of the marked oxidative alterations, the incubation plasma prevented a substantial hemolysis, even after a 18 u h incubation. On the contrary, the exposure of RBCs to 50 u w M MDA in glucose-containing phosphate saline buffer, resulted in a 16% hemolysis within 6 u h. These results indicate that the exposure to MDA causes a rapid intracellular oxidative stress and potentiates oxidative cascades on RBCs, resulting in their dysfunction.

Retinoid dynamics in chicken eye during pre-and postnatal development

Changes in the steady state level of retinols, retinaldehydes and retinyl esters in the trans and 11-cis forms and trans retinoic acid were measured in whole chicken eye during development from day 6in ovo to day 3 post-hatch. These retinoids, quantified by different HPLC systems, were detected in this time sequence: trans-retinol and trans-retinyl esters in the first weekin ovo, 11-cis-retinol in the second week. The highest level of 11-cis-retinaldehyde and 11-cis-retinyl esters was reached at the end of developmentin ovo; however, their levels increased further after hatching. The retinoic acid level decreased at the end of the first week, rising again at the end of the second week.The enzyme activities involved in the metabolism of these retinoids-acyl-CoA: retinol acyltransferase, trans-retinol dehydrogenase, 11-cis-retinol dehydrogenase, trans-retinyl ester hydrolase and trans: 11-cis-retinol isomerase were also estimated and they were detectable already in the first week of developmentin ovo.At day 6 of the biosynthesis of retinoic acid by the retinaldehyde dehydrogenase activity from retina cytosol was also shown.

Retinyl ester hydrolases in retinal pigment epithelium

In bovine retinal pigment epithelium membranes we have found three hydrolases which were active against trans-retinyl palmitate. This was possible by assaying different subcellular fractions as a function of pH in the range 3-9. Detection of these activities has been favored by the use in the enzyme assay of Triton X-100, which has an activating effect up to a concentration of 0.03% at a detergent-protein ratio of about 1.5-3.0. Apparent kinetic parameters for the retinyl ester hydrolases have been determined after a study of the optimization of assay conditions. Vmax values for hydrolases acting at pH 4.5, 6.0, and 7.0 were, respectively, 156, 55, and 70 nmol/h/mg. To identify the subcellular site for these hydrolytic activities, assays of marker enzymes from various organelles in each subcellular preparation were carried out, demonstrating the lysosomal origin of the pH 4.5 retinyl ester hydrolase and the microsomal origin of the pH 6.0 retinyl ester hydrolase and suggesting that the pH 7.0 retinyl ester hydrolase originates from the Golgi complex.

Short-term cactus pear [Opuntia ficus-indica (L.) Mill] fruit supplementation ameliorates the inflammatory profile and is associated with improved antioxidant status among healthy humans

Background Dietary ingredients and food components are major modifiable factors protecting immune system and preventing the progression of a low-grade chronic inflammation responsible for age-related diseases. Objective Our study explored whether cactus pear (Opuntia ficus-indica, Surfarina cultivar) fruit supplementation modulates plasma inflammatory biomarkers in healthy adults. Correlations between inflammatory parameters and antioxidant status were also assessed in parallel. Design In a randomised, 2-period (2 weeks/period), crossover, controlled-feeding study, conducted in 28 healthy volunteers [mean age 39.96 (±9.15) years, BMI 23.1 (±1.5) kg/m2], the effects of a diet supplemented with cactus pear fruit pulp (200 g, twice a day) were compared with those of an equivalent diet with isocaloric fresh fruit substitution. Results With respect to control, cactus pear diet decreased ( p < 0.05) the pro-inflammatory markers such as tumour necrosis factor-α (TNF-α), interleukin (IL)-1β, interferon-γ (INF)-γ, IL-8, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), whereas it increased ( p < 0.05) the anti-inflammatory marker IL-10. Moreover, the diet decreased ratios between pro-inflammatory biomarkers (CRP, IL-6, IL-8, TNF-α) and anti-inflammatory biomarker (IL-10) ( p < 0.05). Cactus pear supplementation caused an increase ( p < 0.05) in dermal carotenoids (skin carotenoid score, SCS), a biomarker of the body antioxidant status, with correlations between SCS and CRP (r = −0.905, p < 0.0001), IL-8 (r = −0.835, p < 0.0001) and IL-10 (r = 0.889, p < 0.0001). Conclusions The presently observed modulation of both inflammatory markers and antioxidant balance suggests cactus pear fruit as a novel and beneficial component to be incorporated into current healthy dietary habits.

Plasma redox response of Sicilian Opuntia Ficus Indica juice in young physically active women

It is known amateur female athletes show an altered redox status [1] and the consumption of Opuntia Ficus Indica (OFI) decreases oxidative stress (OS) in healthy humans [2]. Therefore, the aim of this study was to investigate whether the supplementation with Sicilian OFI juice affected plasma redox balance following a maximal effort test in young physically active women
 . This study was randomized, double blind, placebo controlled and crossover design. Eight women (23.25±2.95 years old, weight of 54.13±9.05 kg, height of 157.75±0.66 cm and BMI of 21.69±0.66 kg/m2) were randomly divided into 2 groups and each group was supplied with either 50 ml OFI, diluted to 170 ml with water, or 170 ml Placebo containing the same concentration of fruit juice ingredients except for Vitamin C and indicaxanthin. They consumed OFI or Placebo every day for 3 days before of effort test on cycle ergometer and continued to take it for 2 consecutive days after testing. Blood samples were taken before and after the effort test without supplementation (baseline), pre- and post-test, 24 h and 48 h post-test with OFI or Placebo supplementation. H2O2 levels and total antioxidant capacity (PAT) were measured with photometer and resonance Raman spectroscopy [1,2]. The differences within and between groups were calculated with ANOVA analysis and considered significant with P<0.05. OFI group showed a significant lower quantity of H2O2 than Placebo group after the effort test. PAT levels of OFI group were significantly higher than pre/post those of baseline and 48 h post-test of Placebo group. In conclusion, OFI supplementation might to be used to restore redox balance after intense exercises in moderately trained women.