Newer strategies for anemia prevention in hemodialysis.

Anemia prevention for hemodialysis relies primarily on supplemental erythropoietin (EPO) and intravenous iron (IV-iron). The doses of EPO utilized are somewhat higher than normal endogenous rates of EPO production in healthy subjects, and the amount of IV-iron used to boost red blood cell (RBC) production may be greater than the amounts used for erythropoiesis. EPO and IV-iron might be used more efficiently if two fundamental problems were solved in the management of dialysis patients: better vitamin C status, and avoidance of chronic inflammation. The low levels of plasma vitamin C commonly observed in dialysis patients restrict mobilization of stored iron from the reticuloendothelial system (RES), and inflammation has a very similar effect. The impact of low vitamin C levels and concurrent inflammation causes a large amount of iron to be stored, with relatively inefficient utilization for erythropoiesis. Vitamin C intake for dialysis patients is often restricted because of avoidance of vitamin C-rich foods, and because of concerns about oxalosis. Inflammation is a chronic feature of renal disease, which is compounded by infections from use of catheters. Research strategies to improve vitamin C status and to decrease inflammation would lead to better utilization of iron and EPO, and could have parallel benefits for the long-term health of patients on hemodialysis.

Methods for measuring ethane and pentane in expired air from rats and humans.

Numerous studies in animals and humans provide evidence that ethane and pentane in expired air are useful markers of in vivo lipid peroxidation. The measurement of breath hydrocarbons, being noninvasive, is well suited for routine use in research and clinical settings. However, the lack of standardized methods for collecting, processing, and analyzing expired air has resulted in the use of a wide variety of different methods that have yielded highly disparate results among investigators. This review outlines the methods that we have developed and validated for measuring ethane and pentane in expired air from rats and humans. We describe the advantages of these methods, their performance, as well as potential errors that can be introduced during sample collection, concentration, and analysis. A main source of error involves contamination with ambient-air ethane and pentane, the concentrations of which are usually much greater and more variable than those in expired air. Thus, it appears that the effective removal of ambient-air hydrocarbons from the subject's lungs before collection is an important step in standardizing the collection procedure. Also discussed is whether ethane or pentane is a better marker of in vivo lipid peroxidation.

Cytosolic and mitochondrial systems for NADH- and NADPH-dependent reduction of alpha-lipoic acid.

In cellular, tissue, and organismal systems, exogenously supplied alpha-lipoic acid (thioctic acid) has a variety of significant effects, including direct radical scavenging, redox modulation of cell metabolism, and potential to inhibit oxidatively-induced injury. Because reduction of lipoate to dihydrolipoate is a crucial step in many of these processes, we investigated mechanisms of its reduction. The mitochondrial NADH-dependent dihydrolipoamide dehydrogenase exhibits a marked preference for R(+)-lipoate, whereas NADPH-dependent glutathione reductase shows slightly greater activity toward the S(-)-lipoate stereoisomer. Rat liver mitochondria also reduced exogenous lipoic acid. The rate of reduction was stimulated by substrates which increased the NADH content of the mitochondria, and was inhibited by methoxyindole-2-carboxylic acid, a dihydrolipoamide dehydrogenase inhibitor. In rat liver cytosol, NADPH-dependent reduction was greater than NADH, and lipoate reduction was inhibited by glutathione disulfide. In rat heart, kidney, and brain whole cell-soluble fractions, NADH contributed more to reduction (70-90%) than NADPH, whereas with liver, NADH and NADPH were about equally active. An intact organ, the isolated perfused rat heart, reduced R-lipoate six to eight times more rapidly than S-lipoate, consistent with high mitochondrial dihydrolipoamide dehydrogenase activity and results with isolated cardiac mitochondria. On the other hand, erythrocytes, which lack mitochondria, somewhat more actively reduced S- than R-lipoate. These results demonstrate differing stereospecific reduction by intact cells and tissues. Thus, mechanisms of reduction of alpha-lipoate are highly tissue-specific and effects of exogenously supplied alpha-lipoate are determined by tissue glutathione reductase and dihydrolipoamide dehydrogenase activity.

Characterization of products formed during the autoxidation of beta-carotene.

The anticarcinogenic action of carotenoids such as beta-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. beta-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 beta-apo-13-carotenone, retinal, beta-apo-14'-carotenal, beta-apo-12'-carotenal, and beta-apo-10'-carotenal. Several very hydrophobic compounds were formed, which have not been previously identified. In addition, the products of NaOCl-treatment of beta-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 beta-carotene in vivo.

Relative reactivity of lysine and other peptide-bound amino acids to oxidation by hypochlorite.

Antibacterial and inflammatory responses of neutrophils and macrophages produce hypochlorite as a major oxidant. Numerous side chains of amino acids found in extracellular proteins can be modified by hypochlorite, including His, Arg, Tyr, Lys, Trp, and Met. We studied the relative reactivity of each of these amino acid residues in short N-blocked peptides, where other residues in the peptide were highly resistant to hypochlorite attack. Hypochlorite treatment led to modified peptides in each case, which were detected by changes in retention on reversed-phase HPLC. A distinct single product, consuming two equivalents of hypochlorite per equivalent of peptide, was obtained from the Lys-containing peptides. UV spectroscopy, nuclear magnetic resonance (NMR), and electrospray/mass spectroscopy identified this product as the dichloramine at the epsilon-amino group of the Lys side chain. The dichloramine at Lys did not decompose to form a detectable amount of carbonyl reactive with dinitrophenylhydrazine. The dichloramine at Lys did however quantitatively revert back to Lys during HCl digestion of the tetrapeptide for amino acid analysis, with simultaneous modification of the adjacent Phe residue. The formation of the dichloramine at Lys was not blocked by peptides or acetylated amino acids that contained Tyr, His, or Arg. In contrast, the presence of equimolar Met-containing peptide, or N-Acetyl-Trp, both inhibited the formation of the dichloramine at Lys. Thus, Met and Trp side chains of proteins might be able to protect Lys from chloramine formation under some circumstances, but this interpretation must consider that Met and Trp are typically found in relatively inaccessible hydrophobic sites, whereas lysine is typically exposed on the protein surface. The hierarchy of amino acid reactivities examined here will aid in the prediction of residues in biological samples most likely to be modified by hypochlorite.

Destruction of tocopherols, carotenoids, and retinol in human plasma by cigarette smoke.

The mechanisms by which exposure to cigarette smoke dramatically increase the incidence and severity of atherosclerosis and the incidence of lung cancer, chronic obstructive airways disease, and emphysema are incompletely understood. Epidemiologic evidence has suggested a modifying role for antioxidant micronutrients, including tocopherols and carotenoids, in these disease processes. It has been suggested that oxidants in cigarette smoke could be involved. We exposed freshly obtained human plasma to the gas phase of cigarette smoke to assess its effects on tocopherols, carotenoids, and retinol. Exposure to cigarette smoke led to the depletion of most of the lipophilic antioxidants in 20 mL human plasma. The order of disappearance was lycopene > alpha-tocopherol > trans-beta-carotene++ > (lutein + zeaxanthin) = cryptoxanthin > gamma-tocopherol = retinol. However, despite a substantial loss of alpha-tocopherol, there was very little peroxidative damage to lipids, and no detectable change in the content of polyunsaturated fatty acid-rich cholesterol esters. We conclude that a wide spectrum of lipophilic micronutrients undergo degradation when exposed to gas-phase cigarette smoke. The relevance of these in vitro findings to possible cigarette smoke-induced depletions of respiratory tract lipophilic antioxidants remains to be clarified.

Human adipose alpha-tocopherol and gamma-tocopherol kinetics during and after 1 y of alpha-tocopherol supplementation.

Alpha-tocopherol and gamma-tocopherol were monitored in human adipose by using needle biopsies in four subjects during a 1-y supplementation trial with 800 mg all-rac-alpha-tocopherol/d, and for 1 additional year after cessation of supplement. Some increase in adipose alpha-tocopherol (per milligram adipose cholesterol) and a more consistent decrease in gamma-tocopherol were observed during the supplementation period. The alpha-tocopherol/gamma-tocopherol ratio rose consistently during supplementation and fell only gradually after the supplement was stopped. We estimate that > or = 2 y are required for the alpha-tocopherol/gamma-tocopherol ratio to reach a new steady state after a change in alpha-tocopherol intake. In a cross-sectional measurement in five subjects who reported long-term use of alpha-tocopherol supplements (> or = 250 mg/d), and in five other subjects who reported no supplement use, the adipose alpha-tocopherol/gamma-tocopherol ratio clearly discriminated between the two groups (P < 0.002). This ratio may be of value in ranking individuals according to long-term alpha-tocopherol intake.

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.

Lutein and zeaxanthin concentrations in plasma after dietary supplementation with egg yolk.

BACKGROUND: The food matrix in which carotenoids are found affects their bioavailability. Lutein and zeaxanthin are abundant in egg yolks and accumulate in the macular region of the retina, where they may affect visual function. OBJECTIVE: We sought to determine whether plasma lutein and zeaxanthin concentrations are elevated after dietary supplementation with egg yolk. DESIGN: Eleven moderately hypercholesterolemic men and women consumed 2 separate baseline diets, which contained 29-33% of energy as total fat, with 20% of energy as either beef tallow or corn oil. These diets were supplemented with cooked chicken egg yolks (1.3 egg yolks/d for an intake of 10.4 MJ). Each subject consumed all 4 diets. Each diet was consumed for 4.5 wk, with a washout period of >/=2 wk between diet phases. At the end of each diet phase, fasting morning plasma samples were collected and stored for carotenoid analysis by HPLC. Commercial chicken egg yolks were analyzed for carotenoids and cholesterol. RESULTS: Egg yolk supplementation of the beef tallow diet increased plasma lutein by 28% (P < 0.05) and zeaxanthin by 142% (P < 0.001); supplementation of the corn oil diet increased plasma lutein by 50% (P < 0.05) and zeaxanthin by 114% (P < 0.001). Changes in plasma lycopene and beta-carotene were variable, with no consistent trend. Egg yolk supplementation increased plasma LDL-cholesterol concentrations by 8-11% (P < 0.05). CONCLUSIONS: Egg yolk is a highly bioavailable source of lutein and zeaxanthin. The benefit of introducing these carotenoids into the diet with egg yolk is counterbalanced by potential LDL-cholesterol elevation from the added dietary cholesterol.

Plasma kinetics of an oral dose of [2H4]retinyl acetate in human subjects with estimated low or high total body stores of vitamin A.

The deuterated retinol dilution technique is an indirect method for quantitatively estimating total body stores of vitamin A by using the postequilibration plasma isotopic ratio [2H4]retinol:retinol and the prediction model described by Furr et al (Am J Clin Nutr 1989;49:713-6). Limited data are available on the time required for an oral dose of labeled vitamin A to mix with vitamin A body stores in human subjects. This article describes the plasma retinol kinetics of an oral dose of [2H4] retinyl acetate in 4 healthy adults (2 men and 2 women) and 1 healthy female child in the United States and in 4 Bangladeshi women. After an oral dose of [2H4]retinyl acetate was administered, plasma samples were collected at 6, 12, and 24 h postdose during the first day and at 15 time points during the subsequent 90-d period for measurement of plasma [2H4]retinol:retinol. The mean respective plasma isotopic ratios on day 20 for US and Bangladeshi subjects (0.02 +/- 0.02 and 0.17 +/- 0.12, P = 0.03) and estimated total body vitamin A reserves (1.03 +/- 0.45 and 0.10 +/- 0.11 mmol, P = 0.003) were significantly different. The fraction of dose in plasma was plotted against time, and biexponential equations were fit to the kinetic data by using the time points from 24 h through day 90. The mean equilibration time (time required for the fraction of dose in plasma to reach a plateau) for all subjects was 16.6 +/- 3.8 d (11-23 d). There was no difference in estimated equilibration time between the group of US and Bangladeshi adult subjects (17.5 +/- 4.4 and 16.3 +/- 3.9 d, respectively, P = 0.69). Thus, the size of hepatic vitamin A reserves does not appear to affect equilibration time within the range of values observed.

Comparison of plasma alpha and gamma tocopherol levels following chronic oral administration of either all-rac-alpha-tocopheryl acetate or RRR-alpha-tocopheryl acetate in normal adult male subjects.

Vitamin E was administered orally (400 IU twice a day) to adult male humans for 28 days as either dl-alpha-tocopheryl acetate (all-rac-alpha-tocopheryl acetate) or d-alpha-tocopheryl acetate (RRR-alpha-tocopheryl acetate). Plasma alpha-tocopherol rose rapidly and fell at the same rate following cessation of supplementation with both forms of vitamin E. No significant differences in plasma alpha- or gamma-tocopherol levels were found between the two forms of vitamin E following their administration. The results confirm the currently accepted biopotencies of 1.0 IU/mg and 1.36 IU/mg, respectively for the two forms of vitamin E. Supplementation with either form of alpha-tocopheryl acetate resulted in depressing plasma gamma-tocopherol to less than 1/3 of initial levels; also the gamma/alpha ratio was depressed to less than 1/7 of the initial value. The study suggests that the gamma/alpha vitamin E ratio might also serve as a sensitive index of alpha-tocopherol ingestion.

Effect of dietary supplementation with black currant seed oil on the immune response of healthy elderly subjects.

BACKGROUND: We have shown that the age-associated increase in prostaglandin E(2) production contributes to the decline in T cell-mediated function with age. Black currant seed oil (BCSO), rich in both gamma-linolenic (18:3n-6) and alpha-linolenic (18:3n-3) acids, has been shown to modulate membrane lipid composition and eicosanoid production. OBJECTIVE: Our objectives were to 1) test whether dietary supplementation with BCSO can improve the immune response of healthy elderly subjects, and 2) determine whether the altered immune response is mediated by a change in the factors closely associated with T cell activation. DESIGN: A randomized, double-blind, placebo-controlled (soybean oil) study was conducted to examine the effect of 2 mo of BCSO supplementation on the immune response of 40 healthy subjects aged >/=65 y. In vivo immune function was determined by delayed-type hypersensitivity skin response. Peripheral blood mononuclear cells (PBMCs) were tested for in vitro immune response. RESULTS: In subjects supplemented with BCSO, the total diameter of induration at 24 h and individual responses to tetanus toxoid and Trichophyton mentagrophytes were significantly higher than their baseline values. The change in response to tetanus toxoid was significantly different from that of the placebo group. The BCSO group showed a significant increase in proliferative response of PBMCs to the T cell mitogen phytohemagglutinin that was not significantly different from that observed in the placebo group. BCSO had no effect on concanavalin A-induced mitogenic response, interleukin 2 and -1beta production, and PBMC membrane fluidity. Prostaglandin E(2) production was significantly reduced in the BCSO-supplemented group, and this change was significantly different from that of the placebo group. CONCLUSION: BCSO has a moderate immune-enhancing effect attributable to its ability to reduce prostaglandin E(2) production.

Assessment of vitamin A status by the deuterated-retinol-dilution technique and comparison with hepatic vitamin A concentration in Bangladeshi surgical patients.

Hepatic stores of vitamin A were estimated in 31 Bangladeshi surgical patients (15 males and 16 females) by the deuterated-retinol-dilution (DRD) technique and by analysis of the vitamin A concentration of a liver biopsy specimen obtained during previously scheduled abdominal surgery. Patients ranged in age from 21 to 65 y and had an average body mass index (BMI: in kg/m2) of 17.7 +/- 3.4. They received 0.753 mumol [2H4]retinyl acetate/kg body wt orally 9-11 d before surgery. Hepatic vitamin A reserves were estimated according to Furr et al (Am J Clin Nutr 1989;49:713-6) by using a single plasma isotopic-ratio measurement (18-25 d postdose). Estimated mean hepatic vitamin A stores were similar by both techniques, 0.110 +/- 0.072 mmol (by DRD) compared with 0.100 +/- 0.067 mmol (by biopsy). Regression analysis was used to compare results of the DRD and biopsy techniques. A significant linear relation was found between the two techniques (r = 0.75, P < 0.0001), and the least-squares regression line was not significantly different from y = x (P = 0.09). The results indicate that the DRD technique provided a very good estimate of hepatic vitamin A reserves for this population. However, a wide prediction interval was observed for estimates of hepatic vitamin A reserves for individual subjects. Thus, further refinement of the prediction model is necessary to improve estimates of hepatic vitamin A reserves for individual subjects.

High-performance liquid chromatography analysis of cholesterol linoleate hydroperoxide in oxidized low density lipoproteins: calibration by conjugated diene internal standard.

The cholesterol linoleate hydroperoxides formed in LDL after oxidant stress are measured by HPLC, with UV detection at 234 nm. Calibration is performed with a conjugated diene internal standard. This internal standard is synthesized by the transesterification of the methyl ester of conjugated diene linoleic acid with a long-chain alcohol, such as arachidyl alcohol (C20). Different long-chain alcohols can be used during the transesterification, to achieve internal standards with variable HPLC retention times. The method allows measurement of cholesterol linoleate hydroperoxide in LDL very early during attack with Cu2+ or other initiator, so that the kinetics of antioxidant loss and hydroperoxide formation can be concurrently monitored.

Analysis of reduced and oxidized lipoic acid in biological samples by high-performance liquid chromatography.

Using HPLC with electrochemical detection at a dual Hg/Au electrode, both reduced and oxidized lipoic acid can be measured in biological samples after addition of lipoic acid. The method does not detect bound lipoic acid, which must be liberated by strong acid or base hydrolysis. The detection limit for this HPLC method is 0.01 nmol of dihydrolipoate and 0.05 nmol of lipoate. Baseline separation of lipoate and dihydrolipoate is achieved on a 10 cm octadecyl column. The analysis is rapid (8 min/sample) and uses a single HPLC pump with isocratic mobile phase. The method has been adapted to study cellular and whole animal reduction of lipoate, membrane transport of lipoate and dihydrolipoate, and subcellular enzymes that reduce lipoate.

Distribution of individual macular pigment carotenoids in central retina of macaque and squirrel monkeys.

The spatial distribution of lutein (L) and zeaxanthin (Z), the structural isomers composing the macular pigment, was studied in the retinas of macaque monkeys (Macaca fascicularis) and squirrel monkeys (Saimiri sciureus). Spatial profiles of macular pigment optical density were obtained from retinal whole mounts. Then concentric annuli were microdissected from the fovea and adjacent regions of the same retinas. Each retinal segment was analyzed for carotenoids by high-performance liquid chromatography. Both L and Z reached their highest concentrations at the center of the fovea and declined monotonically with eccentricity for both primate species. This is inconsistent with a preferential association of L with rods. Macaque monkeys have a consistent pattern of more Z than L at the foveal center, like humans. Z declines more rapidly than L with eccentricity, so that L becomes dominant in the periphery. Squirrel monkeys (all male) showed striking individual differences. Some had more Z than L at the foveal center like macaques, but four of six had the reverse pattern, with more L than Z throughout the central retina. Individual differences among squirrel monkeys may be linked to their color vision polymorphisms. This suggests that a particular Z/L ratio in primate retinas may be associated with a specific cone phenotype, just as particular carotenoids are associated with specific cone types in vertebrates with cone oil droplets.

Carotenoids in the human macula and whole retina.

The carotenoid pigments in the whole human retina and in the macular region were measured quantitatively by high pressure liquid chromatography (HPLC). Approximately a five-fold larger amount of carotenoids was found in the human macula (35-120 ng) than in previously reported work. The dominant carotenoids in the whole retina are lutein and zeaxanthin. Zeaxanthin is concentrated in the macular region, whereas lutein is dispersed throughout the entire retina. Contrary to prior reports, substantial quantities of both carotenoids are present in the infant retina. Increasing variability is observed in carotenoid levels between individuals with advancing age, and some older individuals show very high whole retina carotenoid levels. These quantitative studies were made possible by synthesis of a new, stable carotenoid internal standard. Carotenoids have been proposed to be potent antioxidants, protecting membrane lipids from toxic peroxidation reactions. The method presented in this study will facilitate quantitative investigations of the association between carotenoid levels and health and disease of the retina.

Biological control of primate macular pigment. Biochemical and densitometric studies.

The amounts of zeaxanthin (Z) and lutein (L), the carotenoids constituting the primate macular pigment, were measured in the central retinas of monkeys (Saimiri sciureus and Macaca fascicularis). Two independent methods--reverse-phase high-performance liquid chromatography (HPLC) and microdensitometry--were used for analysis of the same set of retinas. Most of the measurements were made on retinas that had been fixed by glutaraldehyde-paraformaldehyde perfusion of the animal. Control experiments showed that this fixation did not interfere with the quantitative extraction and analysis of the carotenoids. The amount of macular pigment calculated from microdensitometry of the foveal region was proportional to the amount of pigment assayed by HPLC of the same retinal area, demonstrating that either method can be used reliably to rank the carotenoid content of aldehyde-fixed foveas. The optical density of pigment in the axial direction through the retina was higher than would be predicted if the pigment were randomly oriented. This is consistent with the idea that the nonrandom orientation of the dichroic macular pigment molecules found in previous studies contributes to increased optical filtering of the retinal image. Comparisons of the amounts of Z and L between the left and right eyes of the same monkey, within 1 mm of the foveal center, always showed excellent agreement (averaging a 5% difference for Z and 11% difference for L), whereas differences among individual monkeys were very large (up to fourfold for Z). These results indicate that the uptake and assimilation of the macular carotenoids are biologically regulated by selective mechanisms in primate retinas.

Iron-induced fluorescence in the retina: dependence on vitamin A.

PURPOSE: Intravitreal iron injection induces fluorophore formation in the photoreceptor outer segments, followed by an accumulation of inclusions with lipofuscin-like fluorescence in the retinal pigment epithelium (RPE). The accumulation of RPE lipofuscin during aging is dependent on vitamin A availability. Experiments were conducted to determine whether iron-induced fluorophore formation in the outer segments and in RPE is also dependent on vitamin A, and thus whether oxidation promotes the participation of vitamin A in lipofuscin formation. METHODS: For 23 weeks, beginning at weaning, albino Fischer rats were fed diets containing vitamin A either in the form of retinyl palmitate (+A), which can be metabolically converted into the retinoids involved in vision, or retinoic acid (-A), which does not support visual function. After 23 weeks, when rhodopsin levels had decreased more than 90% in the -A rats, some animals in this group were given an intramuscular injection of all-trans retinol and were allowed to recover from retinoid deficiency for 7 days (-A+A). Animals in all three treatment groups were then given an intravitreal injection of ferrous sulfate. Both 1 day and 7 days after the iron injections, the retinas and RPEs were examined for fluorophores with excitation and emission properties similar to those of RPE lipofuscin fluorophores. RESULTS: In retina sections examined with fluorescence microscopy 24 hours after the ferrous sulfate treatment, the photoreceptor outer segments of rats in all of the treatment groups displayed a fluorescence with a blue emission maximum. This outer-segment fluorescence was not present in untreated eyes. The in situ outer-segment fluorescence was correlated with the appearance of blue-emitting fluorophores in organic solvent extracts of the retinas. One week after the iron injections, the RPE cells of the +A animals became filled with inclusions that displayed a golden-yellow fluorescence emission when excited by blue light. Very little of this lipofuscin-like fluorescence was observed in the RPE of the -A rats 1 week after iron treatment. However, in the -A rats that had been repleted with vitamin A, the ability of iron to induce the RPE fluorescence was restored. Several orange-emitting fluorophores were present in organic solvent extracts of the RPE-choroids of the +A rats. The amounts of these fluorophores were not appreciably affected by the iron treatment. These orange-emitting compounds were not observed in extracts of any eyes in the -A or -A+A groups. CONCLUSIONS: The results of this study suggest that oxidation of the photoreceptor outer-segment lipids generates blue-emitting fluorophores that are not directly involved in RPE lipofuscin fluorophore formation. The findings also indicate that retinoids are direct precursors of RPE lipofuscin fluorophores, and that oxidative stress to the retina promotes participation of vitamin A in the formation of some of the compounds responsible for RPE lipofuscin fluorescence.

Oxidative stress and inflammation in hemodialysis patients.

Dialysis is associated with an increased generation of oxidants, which play an important part in the development of endothelial dysfunction and atherogenesis. Markers of oxidative stress include F2-isoprostanes and ethane. Measurements in dialysis patients before dialysis showed higher levels of esterified plasma F2-isoprostanes (1.62 +/- 0.73 ng/mL) than in control subjects (0.27 +/- 0.10 ng/mL) (P < 0.001). Furthermore, levels also correlated with high plasma C-reactive protein (CRP) levels (r =.48, P = 0.015). Breath ethane levels for dialysis patients (N = 19) were 6.32 +/- 3.16 pmol/kg-min, in contrast to 3.08 +/- 1.50 pmol/kg-min in control subjects (N = 11, P < 0.005). Analysis to investigate the relationship between CRP levels and outcome indicated that there was a significant difference in mortality rate over a 3-year period between patients with low and high CRP values (P < 0.001). Patients with high CRP (> 16.8 mg/L) levels were more than twice as likely to die as patients with low CRP levels (relative risk [RR] = 2.16; 95% confidence interval [CI], 1.50-3.09). CRP values were a significant predictor of mortality even after controlling for diabetes, albumin, ferritin, and age at commencement of dialysis. The RR for CRP after adjustment was 1.58 (95% CI, 1.06-2.34, P = 0.024). There were no significant interactions between CRP and other predictors of mortality, indicating that high CRP levels have an additive effect on the mortality risk. These findings show that hemodialysis patients are exposed to both oxidative stress and inflammation.