Garry J. Handelman

A new role for phospholipase A2: protection of membranes from lipid peroxidation damage

Abstract Recently it was discovered that phospholipase A 2 preferentially hydrolyses peroxidized fatty acid esters in phospholipid membranes. Release of the peroxidized fatty acids from the membrane was found to be an absolute requirement for glutathione peroxidase to reduce and detoxify fatty acid hydroperoxides in membranes. On the basis of these findings we propose a new role for phospholipase A 2 in protecting membranes from oxidative injury.

Dietary antioxidants and cigarette smoke-induced biomolecular damage: a complex interaction.

Epidemiologic evidence suggests that cigarette smoking is a major risk factor for chronic obstructive pulmonary diseases such as chronic bronchitis and emphysema, for carcinogenesis, and for cardiovascular disease. However, the precise mechanisms of these effects are incompletely understood. The gas phase of cigarette smoke contains abundant free radicals including nitric oxide. Hence, cigarette smoke may induce some of its damaging effects by free radical mechanisms. We report that exposure of plasma, a model for respiratory tract lining fluids, to gas-phase cigarette smoke causes depletion of antioxidants, including ascorbate, urate, ubiquinol-10, and alpha-tocopherol, and a variety of carotenoids, including beta-carotene. Gas-phase cigarette smoke induced some lipid peroxidation, as measured by cholesteryl linoleate hydroperoxide (18:2OOH) formation. Ascorbate was effective in preventing 18:2OOH formation. In contrast to the low concentrations of lipid hydroperoxides measured (< 1 mumol/L), protein carbonyl formation, a measure of protein modification, increased by approximately 400 mumol/L after nine puffs of cigarette smoke. Reduced glutathione inhibited protein carbonyl formation, whereas other plasma antioxidants, including ascorbate, were ineffective. alpha, beta-Unsaturated aldehydes (acrolein and crotonaldehyde) in cigarette smoke may react with protein -SH and -NH2 groups by a Michael addition reaction that results in a protein-bound aldehyde functional group. Gas-phase cigarette smoke is capable of converting tyrosine to 3-nitrotyrosine and dityrosine, indicating free radical mechanisms of protein damage by nitrogen oxides. Aldehydes and nitrogen oxides in cigarette smoke may be significant contributors to biomolecular damage, and endogenous antioxidants can attenuate some of these adverse effects.

Characterization of products formed during the autoxidation of β-carotene

Abstract The anticarcinogenic actions of carotenoids such as β-carotene has been frequently ascribed to their antioxidant properties. However, very little is actually known about the nature of the antioxidant reaction or the products that are formed. β-Carotene was exposed to either spontaneous autoxidation conditions or to radical-initiated autoxidation conditions. The products were separated by reverse-phase HPLC, and individual peaks were characterized with an on-line diode array detector. Carbonyl products were isolated and characterized by several procedures, including borohydride reduction to the corresponding alcohols, derivatization with O -ethyl-hydroxylamine to the corresponding O -ethyl-oximes of the carbonyls, and analysis by GC-MS. Under the conditions of the experiments, the formation of a homologous series of carbonyl products was demonstrated, including β-apo-13-carotene, retinal, β-apo-14′-carotenal, β-apo-12′-carotenal, and β-apo-10′-carotenal. Several very hydrophobic compounds were formed, which have not been previously identified. In addition, the products of NaOCl-treatment of β-carotene were analyzed, and shown to be significantly different from the autoxidation products. This type of product analysis should be useful in determining the nature of the oxidants reacting with β-carotene in vivo.

REDOX REGULATION OF UBIQUITIN-CONJUGATING ENZYMES : MECHANISTIC INSIGHTS USING THE THIOL-SPECIFIC OXIDANT DIAMIDE

The ubiquitin–proteasome pathway (UPP) regulates critical cell processes, including the cell cycle, cytokine‐induced gene expression, differentiation, and cell death. Recently we demonstrated that this pathway responds to oxidative stress in mammalian cells and proposed that activities of ubiquitin‐activating enzyme (E1) and ubiquitin‐conjugating enzymes (E2s) are regulated by cellular redox status (i.e., GSSG:GSH ratio). To test this hypothesis, we altered the GSSG:GSH ratio in retinal pigment epithelial cells with the thiol‐specific oxidant, diamide, and assessed activities of the UPP. Treatment of cells with diamide resulted in a dose‐dependent increase in the GSSG:GSH ratio resulting from loss of GSH and a coincident increase in GSSG. Increases in the GSSG:GSH ratio from 0.02 in untreated cells to ≥0.5 in diamide‐treated cells were accompanied by dose‐dependent reductions in the levels of endogenous Ub‐protein conjugates, endogenous E1∼ubiquitin thiol esters, and de novo ubiquitin‐conjugating activity. As determined by the ability to form E1‐ubiquitin and E2s‐ubiquitin thiol esters, E1 and E2s were both inhibited by elevated GSSG:GSH ratios. Inhibition of E1 was associated with the formation of E1‐protein mixed disulfides. Activities of E1 and E2s gradually recovered to preoxidation levels, coincident with gradual recovery of the GSSG:GSH ratio. These data support S‐thiolation/dethiolation as a mechanism regulating E1 and E2 activities in response to oxidant insult. Ubiquitin‐dependent proteolytic capacity was regulated by the GSSG:GSH ratio in a manner consistent with altered ubiquitin‐conjugating activity. However, ubiquitin‐independent proteolysis was unaffected by changes in the GSSG:GSH ratio. Potential adaptive and pathological consequences of redox regulation of UPP activities are discussed.

The evolving role of carotenoids in human biochemistry.

The growth of our knowledge of carotenoid biochemistry has opened new and divergent paths for research. The earliest role established for beta-carotene in animals was as a vitamin A precursor, a role it shares with several other pro-vitamin A carotenoids. Additional studies have continued to refine our understanding of this function. Because carotenoids are excellent scavengers of singlet oxygen and respectable scavengers for other reactive oxygen species, substantial work was done concerning their potential role as antioxidants. In an unexpected twist, the ability of radicals in cigarette smoke to degrade carotenoids might be responsible for the finding that high-dose dietary beta-carotene increased the incidence of lung cancer in smokers. A new role for the polar carotenoids lutein and zeaxanthin was identified, when those carotenoids were found to constitute the macular pigment (the yellow spot at the center of the human retina). Many different carotenoids can be metabolized to products with retinoid activity, which might affect gene expression and cell differentiation. The formation of retinoids from diverse carotenoids might account for a portion of their activities as anticancer agents. Studies of lycopene in prostate cancer prevention have been very promising, and clinical studies of lycopene are underway. Carotenoids have emerged as the best single tissue marker for a diet rich in fruits and vegetables, and measurements of plasma and tissue carotenoids have an important role in defining the optimal diets for humans.

The role of antioxidants in the retina and retinal pigment epithelium and the nature of prooxidant-induced damage

Abstract The vertebrate retina has several features that make it vulnerable to damage from autoxidation. The photoreceptor membranes contain high levels of polyunsaturated fatty acids; abundant mitochondria are present which may leak activated oxygen species; and light exposure of the retina may cause photoxidation. These features are analyzed in detail, and the various antioxidant mechanisms of the vertebrate retina are surveyed. The interplay among oxidant stress and antioxidant defenses is illustrated by review of situations where these variables are either artificially manipulated or changed naturally. Vitamin E deficiency damages the retina in a number of well-defined vertebrate model systems, and a lipid autoxidation mechanism for this damage is widely assumed. The retina is quite sensitive to damage by elevated or prolonged light exposure; however, a free-radical role in light damage to the retina has not been established. An alternative mechanism for damage due to vitamin E deficiency and light is considered, which involves elevated vitamin A levels and vitamin A toxicity. Evidence is reviewed that the primate retina requires both vitamin E and selenium. The puzzling role of ocular melanin in light damage and protection is reviewed. Possible contributions of autoxidative damage to aging of the human retina are discussed.

α-Lipoic acid reduction by mammalian cells to the dithiol form, and release into the culture medium

Lipoic acid has been reported recently to be an effective antioxidant in biological systems. It may act in vivo through reduction to its dithiol form, dihydrolipoic acid. Using a dual Hg/Au electrode, and HPLC with electrochemical detection, a method was developed which allowed simultaneous measurement of lipoic acid and dihydrolipoic acid, at nanomolar levels. (RS)-alpha-Lipoic acid was added to human cells in tissue culture (Jurkat T-lymphocytes and primary neonatal diploid fibroblasts). Lipoic acid was converted rapidly by the cells to dihydrolipoic acid, which accumulated in the cell pellet. Monitored over a 2-hr interval, dihydrolipoic acid was released, and several-fold more dihydrolipoic acid could be found in the medium than in the pellet.

Effects of antioxidant nutrient deficiency on the retina and retinal pigment epithelium of albino rats: a light and electron microscopic study

Abstract Male Sprague-Dawley rats were maintained, from weaning, on diets deficient either in vitamin E, selenium, chromium and sulfur amino acids (−E−Se−S−Cr), or only in vitamin E and selenium (−E−Se+S+Cr). Control animals (+E+Se+S+Cr) received all four nutrients. After 24–26 weeks on their respective diets, both deficient groups showed a dramatic accumulation of an autofluorescent pigment, similar to lipofuscin, in the retinal pigment epithelium. This increased autofluorescence was correlated with a large increase in the number of electron-dense inclusion bodies observed in the pigment epithelium by transmission electron microscopy. Accompanying the build-up of autofluorescent pigment was the development of an irregularity and an overall increase in retinal pigment epithelium cell height. There was also an increase in the number of lipid droplets in the retinal pigment epithelium, particularly in the periphery of the eye. In the eyes of deficient animals, cells were occasionally seen which appeared to have detached from Bruchs membrane and migrated into the region of the photoreceptor outer segments. Photoreceptor outer segment phagocytosis by the retinal pigment epithelium appeared to be decreased as a consequence of dietary antioxidant deficiency, since a reduction of over 75% in the number of phagosomes per unit retinal pigment epithelium cell length was seen in some areas of the eye in both deficient groups. These changes in the retinal pigment epithelium were accompanied by a pronounced loss of photoreceptor cells, particularly from the central retina (20–34% fewer cells than supplemented controls). The disk membranes of the photoreceptor outer segments of deficient animals were often swollen, disoriented and vesiculated, and areas were frequently seen where outer segment debris had accumulated at the interface between the photoreceptors and retinal pigment epithelium. Many of the changes in the retina and retinal pigment epithelium were more severe in the fully deficient (−E−Se−S−Cr) group, than in the group deficient only in vitamin E and selenium.

Iron and anemia in human biology: a review of mechanisms

The biology of iron in relation to anemia is best understood by a review of the iron cycle, since the majority of iron for erythropoiesis is provided by iron recovered from senescent erythrocytes. In iron-deficiency anemia, storage iron declines until iron delivery to the bone marrow is insufficient for erythropoiesis. This can be monitored with clinical indicators, beginning with low plasma ferritin, followed by decreased plasma iron and transferrin saturation, and culminating in red blood cells with low-Hb content. When adequate dietary iron is provided, these markers show return to normal, indicating a response to the dietary supplement. Anemia of inflammation (also known as anemia of chronic disease, or ACD) follows a different course, because in this form of anemia storage iron is often abundant but not available for erythropoiesis. The diagnosis of ACD is more difficult than the diagnosis of iron-deficiency anemia, and often the first identified symptom is the failure to show a response to a dietary iron supplement. Confirmation of ACD is best obtained from elevated markers of inflammation. The treatment of ACD, which typically employs erythropoietin (EPO) supplements and intravenous iron (IV-iron), is empirical and often falls shorts of therapeutic goals. Dialysis patients show a complex pattern of anemia, which results from inadequate EPO production by the kidney, inflammation, changes in nutrition, and blood losses during treatment. EPO and IV-iron are the mainstays of treatment. Patients with heart failure can be anemic, with incidence as high as 50%. The causes are multifactorial; inflammation now appears to be the primary cause of this form of anemia, with contributions from increased plasma volume, effects of drug therapy, and other complications of heart disease. Discerning the mechanisms of anemia for the heart failure patient may aid rational therapy in each case.

Consecutive action of phospholipase A2 and glutathione peroxidase is required for reduction of phospholipid hydroperoxides and provides a convenient method to determine peroxide values in membranes

The purpose of this study was to investigate the ability of selenium-dependent glutathione peroxidase to reduce phospholipid hydroperoxides in membrane bilayers and to develop a method to measure the peroxide content of phospholipids. Phospholipid hydroperoxides were synthesized by photooxidation of 1-palmitoyl 2-linoleoyl phosphatidylcholine and characterized by gas chromatography-mass spectrometry. Phospholipid hydroperoxides in phosphatidylcholine bilayers showed no detectable reactivity with Se-dependent glutathione peroxidase (the reaction is at least 65,000 times slower than with an available hydroperoxide). However, after the phospholipid hydroperoxides were preincubated with phospholipase A2, the free fatty acid hydroperoxides became available as a substrate for Se-dependent glutathione peroxidase. The enzyme assay can be used for convenient determination of peroxide values in phospholipids at the 1 nmole level and free fatty acid hydroperoxides can be distinguished from phospholipid hydroperoxides by omitting phospholipase A2. The accuracy of the enzymatic method was confirmed using an improved colorimetric chemical assay to measure peroxide values of phospholipid hydroperoxides to the same sensitivity. The chemical assay was not linear in the presence of high levels of lipid, but at low levels of lipid the peroxide values of phospholipid hydroperoxides measured by both methods agreed to within 1%. Since high levels of lipid inhibited the chemical assay, the enzyme assay is more accurate for determination of peroxides in membranes and tissues. The possible role of phospholipase deficiencies as a causal factor in degenerative diseases thought to be due to lipid peroxidation, such as Neuronal Ceroid Lipofuscinosis (Battens disease), is discussed.