Antonino Bongiorno

Oral supplements of vitamin E improve measures of oxidative stress in plasma and reduce oxidative damage to LDL and erythrocytes in β-thalassemia intermedia patients

Fifteen β-thalassemia intermedia patients, not requiring chronic transfusional therapy, were monitored in order to check their antioxidant status, and the lipid oxidation products in plasma, LDL, and erythrocytes before and during a 9-month oral treatment with 600 mg/day vitamin E. The low level of vitamin E, and high level of malondialdehyde in plasma clearly tended to normalize after three months (P<.001), and were quite similar to control after six months. The abnormally low level of vitamin E in LDL and the four times higher than control basal level of conjugated dienes (LDL-CD), were not modified after three months of treatment. Significant changes of LDL-VE (P<.05) and of the basal LDL-CD (P<.001) were evident after six months. LDL-VE was within the normal range after nine months, whereas LDL-CD still appeared twice as higher than control. Plasma vitamin A, ascorbate, β-carotene, and lycopene increased markedly at the end of the trial (P<.005). The level of vitamin E in red blood cells was normalized after six months of supplementation. A decrease of the baseline value of conjugated dienes was observed after nine months, although it remained 1.4-fold higher than control. The RBC count and hematocrit appeared higher at the end of the trial (P<.05 and P<.001, respectively). The hemoglobin value did not show variations. A shift to normal of the resistance of erythrocytes to osmotic lysis was observed. Our findings provide evidence that an oral treatment with vitamin E improves the antioxidant/oxidant balance in plasma, LDL particles, and red blood cells, and counteracts lipid peroxidation processes in β-thalassemia intermedia patients.

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.

Distribution of vitamin A compounds in bovine eyes after bleaching adaptation.

A seasonal increase in the amount of bleached rhodopsin caused, in living animals, by the seasonal increase of the intensity of sunlight in the early morning before the calves are killed, was verified in the bovine eyes subjected to the present study. This was used as a means of assaying distribution and isomer composition of esterified and unesterified retinol in eyes from animals light-adapted to a different extent under environmental conditions. The progressive increase of bleached rhodopsin results in a parallel increase of all-trans-retinol in retina and of both all-trans- and 11-cis-retinyl esters in pigment epithelium. Analytical subcellular fractionation of RPE homogenate reveals that retinyl esters accumulate without an exclusive subcellular localization in nuclear, mitochondrial/lysosomal and microsomal fractions. Whatever the amount of bleached rhodopsin, only small and constant amounts of retinyl esters are found in the soluble fraction of RPE, entirely under the all-trans configuration. When a considerable portion of rhodopsin is bleached (about 70%), substantial amounts of all-trans-retinol, along with minor amounts of 11-cis-retinol, accumulate in RPE subcellular organelles. The in vitro bleaching of bovine eyes results in a distribution of retinoids between retina and RPE which appears different from that detected in eyes naturally bleached to the same extent.

Modulation by Docosahexaenoic Acid of the Epinephrine-Stimulated Adenylate Cyclase Activity of the Bovine Retina

Abstract: This work shows that unsaturated fatty acids enhance the epinephrine‐stimulated adenylate cyclase activity in bovine retina. The modulating effect on the epinephrine‐stimulated formation of cyclic AMP seems to be linked to the degree of unsaturation of the fatty acid. Treatment of the intact retina with docosahexaenoic acid in the concentration range 0.5 × 10‐6‐l × 10‐3M does not affect the enzyme activity measured in the absence of the hormone but markedly increases the cyclase activity when the tissue is incubated in the presence of 0.1 mM epineph‐rine. Docosahexaenoic acid enhances the maximal response to epinephrine without affecting the apparent ED50 value for this effector. Docosahexaenoic acid at 0.5 mM also increases the hormone‐stimulated adenylate cyclase activity in retinal cell‐free homogenate, whereas it has no effect on the epinephrine‐sensitive enzyme solubilized from the membrane fraction with 1% Triton X‐305. When docosahexaenoic acid‐preincubated intact retina and cell‐free Homogenate are incubated in the presence of defatted albumin, both the observed activating effect of the fatty acid on the epinephrine‐stimulated adenylate cyclase activity and the enhancement of the enzyme response to the hormone significantly diminish.

All-trans to 11-cis retinol isomerization in nuclear membrane fraction from bovine retinal pigment epithelium

Isomerization of all-trans to 11-cis retinol has been studied in a membrane preparation from the nuclear fraction of bovine retinal pigment epithelium. When the nuclear membrane preparation deprived of endogenous retinoids is incubated with 4.5 microM all-trans-retinol, the mean value calculated for the isomerase activity is 1.32 nmol 11-cis retinol formed hr-1 mg protein-1. Simultaneous formation of all-trans and 11-cis retinyl esters is also observed in the nuclear preparation. When assayed under the same experimental condition, RPE 150,000 g post-nuclear sediment shows about 70% of the isomerase activity found in the nuclear membrane fraction. Treatment of the nuclear membrane fraction with 0.5% (w/v) CHAPS produces a 200,000-g supernatant retaining 80% of the total isomerase activity and leads to a modest purification of the enzyme activity. Apparent values for Km and Vmax of the solubilized enzyme are 1.6 microM and 2.5 nmol 11-cis retinol formed h-1 mg protein-1, respectively. Bovine serum albumin and beta-lactoglobulin effectively stimulate the isomerization reaction. The mechanism underlying this activating effect remains unclarified at present. Some hypotheses are discussed.

Binding of 11-cis retinaldehyde to the partially purified cellular retinaldehyde binding protein from bovine retinal pigment epithelium.

11-cis retinaldehyde binding analysis was performed on a bovine retinal pigment epithelium preparation of cellular retinaldehyde binding protein (CRALBP), whose purity degree was estimated as 75%. Equilibrium binding studies were carried out measuring the replacement of tritium-labeled with unlabeled 11-cis retinaldehyde at 25°C. Analysis of the experimental data both by a direct curve-fitting procedure utilizing a non linear least square regression analysis and by a conventional Scatchard plot revealed a single non-interacting binding site with an apparent equilibrium constant of 0.9×10−7 M. A binding stoichiometry of approximately 1 mol of 11-cis retinaldehyde/mol of binding protein can be calculated from the experimental data. Competition studies carried out in the presence of unlabeled ‘trans’ and ‘cis’ isomers of Vitamin A derivatives confirm the high degree of specificity of the 11-cis retinaldehyde binding.

Analysis of a soluble lipid-protein complex carrying endogenous 11-cis retinaldehyde from bovine retinal pigment epithelium

SummaryA soluble lipid-protein complex in bovine retinal pigment epithelium is shown to carry endogenous 11-cis retinaldehyde, in the extent of 15% of the total 11-cis retinaldehyde found in this tissue. The complex, analyzed with respect to its chemical composition, exhibits a lipid composition close resembling the lipid composition of the rod outer segment membrane; the SDS-PAGE evidences the presence of a number of protein bands, two of which of 34 and 27 kDa appear glycoproteins. Finally, the lipid-protein complex exhibits a discrete level of a Cathepsin D-like protease activity. From the above, the possibility is discussed that the soluble lipid-protein complex could represent some phagolysosomal inclusion occurring in the pigment epithelial cells upon rod outer segment phagocytosis.

Isolation procedure for bovine retinal rod outer segments.

An isolation procedure to obtain rod outer segments from cattle retinas is reported. Centrifugation of homogenates in discontinuous and continuous sucrose density gradients yields purified photoreceptor cell outer segments. Assay of the final preparation for rhodopsin content gives a ratio of 2.4 for DO280 nm/DO498 nm.