Non-protein bound iron release during chemotherapy in cancer patients.

Non-protein bound iron (NPBI) is able to catalyse oxidative reactions, causing damage to vital structures. Adverse effects induced by cisplatin seem, in part, to be mediated by free radicals. In the present study, we have measured plasma NPBI, various other iron parameters and antioxidants in 28 cancer patients undergoing cisplatin-based chemotherapy at various time points before and during chemotherapy. No NPBI was present prior to therapy, but within 1-4 days following the first administration of chemotherapy, mean NPBI rose significantly to 10.6+/-6.6 micromol/l (range, 0.6-21.3 micromol/l) in 18 (64.3%) of the 28 patients measured. The rise in NPBI was accompanied by a significant rise in total plasma iron and ferritin and a marked decrease in the latent iron-binding capacity. Concomitantly, plasma vitamins C and E decreased significantly, indicating consumption of antioxidants. Similar observations were also made during the fourth chemotherapy cycle. The increase in NPBI preceded and correlated significantly with chemotherapy toxicity, such as a decrease in leucocyte count and haemoglobin, with a transient rise in various liver enzymes and with known cisplatin-related toxicity, i.e. the loss of renal and hearing function. In conclusion, cisplatin chemotherapy induces oxidative damage which rapidly leads to release of iron from intracellular proteins and the appearance of NPBI. Bone marrow, red blood cells, liver and kidney seem to be a likely source of NPBI. The observed high levels of NPBI may be a major causative determinant in chemotherapy-induced toxicity.

Increased antioxidant potential of combined topical vitamin E and C against lipid peroxidation of eicosapentaenoic acid in pig skin induced by simulated solar radiation.

PURPOSE: To study the protective effect of topically applied vitamin E (TOC), vitamin C (ASC), or a combination of both, against the lipid peroxidation of eicosapentaenoic acid (EPA) induced by simulated solar radiation (SSR). MATERIAL AND METHODS: EPA (25 nmol cm(-2)) was topically applied to pig skin explants, followed by increasing doses of TOC and ASC, either alone or combined. Epidermal lipid peroxidation was assessed after 15 min of exposure to SSR (resulting in a UVA and UVB dose of 18 and 3 kJ m(-2), respectively). SSR-induced changes in the levels of TOC and ASC were determined in the stratum cornaeum and the viable epidermis. RESULTS: SSR exposure of EPA-treated pig skin resulted in a twofold increase in epidermal lipid peroxidation (p <0.005) which was reduced by topically applied TOC or ASC 60 min before SSR exposure (p <0.05). Compared with TOC (5 nmol cm(-2)), a 400-fold higher ASC dose was needed and only TOC provided complete protection against the lipid peroxidation of EPA. The levels of both TOC and ASC clearly increased in both skin compartments by increasing the applied dose of these two compounds (p <0.05). In contrast to ASC, TOC was consumed by up to 55-70% during SSR exposure (p <0.05). Compared with separate application, combined TOC and ASC efficiently protected against lipid peroxidation of EPA at doses that were five and 200 times lower, respectively. In the presence of low ASC doses, 70-100% of epidermal TOC was regenerated during SSR exposure (p <0.05). CONCLUSIONS: Topically applied TOC and ASC protect against SSR-induced lipid peroxidation of EPA. The synergism between TOC and ASC resulted in a more efficient protection at substantially lower doses of both antioxidants. Co-supplementation of EPA with TOC and/or ASC might improve the beneficial biological effects of EPA.

Dietary eicosapentaenoic acid prevents systemic immunosuppression in mice induced by UVB radiation.

Moison, R. M. W. and Beijersbergen van Henegouwen, G. M. J. Dietary Eicosapentaenoic Acid Prevents Systemic Immunosuppression in Mice Induced by UVB Radiation. Radiat. Res. 156, 36-44 (2001). Reactive oxygen species (ROS) contribute to the immunosuppression induced by UVB radiation. Omega-3 fatty acids in fish oil, e.g. eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), can modulate immunoresponsiveness, but because of their susceptibility to ROS-induced damage, they can also challenge the epidermal antioxidant defense system. The influence of dietary supplementation with different omega-3 fatty acids on systemic immunosuppression induced in mice by UVB radiation was studied using the contact hypersensitivity response to trinitrochlorobenzene. In an attempt to study the mechanisms involved, UVB-radiation-induced changes in epidermal antioxidant status were also studied. Mice received high-fat (25% w/w) diets enriched with either oleic acid (control diet), EPA, DHA, or EPA + DHA (MaxEPA). Immunosuppression induced by UVB radiation was 53% in mice fed the oleic acid diet and 69% in mice fed the DHA diet. In contrast, immunosuppression was only 4% and 24% in mice fed the EPA and MaxEPA diets, respectively. Increased lipid peroxidation and decreased vitamin E levels (P < 0.05) were found in unirradiated mice fed the MaxEPA and DHA diets. For all diets, exposure to UVB radiation increased lipid peroxidation (P < 0.05), but levels of glutathione (P < 0.05) and vitamin C (P > 0.05) decreased only in the mice given fish oil. UVB irradiation did not influence vitamin E levels. In conclusion, dietary EPA, but not DHA, protects against UVB-radiation-induced immunosuppression in mice. The degree of protection appears to be related to the amount of EPA incorporated and the ability of the epidermis to maintain an adequate antioxidant level after irradiation.

Topically applied eicosapentaenoic acid protects against local immunosuppression induced by UVB irradiation, cis-urocanic acid and thymidine dinucleotides.

UVB-induced immunosuppression, a promoter of photocarcinogenesis, involves the formation of pyrimidine dimers and cis-urocanic acid (cis-UCA), but reactive oxygen species (ROS) also plays an important role. Eicosapentaenoic acid (EPA) can inhibit photocarcinogenesis, but due to its polyunsaturated nature it is susceptible to oxidative damage by ROS. The antioxidant defense system may therefore be challenged upon ultraviolet-B (UVB) irradiation in the presence of EPA. We investigated whether topically applied EPA in mice could protect against local immunosuppression (contact hypersensitivity response to dinitrofluorobenzene) induced by UVB radiation (1.5 J/cm2), or topically applied cis-UCA (150 nmol/cm2) or thymidine dinucleotides (pTpT) (5 nmol/cm2). The influence of EPA on epidermal lipid peroxidation and antioxidant status was also measured. UVB irradiation, cis-UCA and pTpT all caused 70% immunosuppression. Topical pretreatment of mice with EPA partially protected against immunosuppression; the EPA dose needed to accomplish this was 10 nmol/cm2 for UVB irradiation, 100 nmol/cm2 for cis-UCA and 1000 nmol/cm2 for pTpT. Higher EPA doses caused higher UVB-induced lipid peroxidation and lower vitamin C levels. Glutathione only decreased with the highest EPA dose whereas vitamin E was not decreased after UVB irradiation. In conclusion, topically applied EPA protects against UVB-, cis-UCA- and pTpT-induced immunosuppression and maintenance of an adequate antioxidant defense seems to be an important prerequisite for the protective action by EPA.

The capacity of different infusion fluids to lower the prooxidant activity of plasma iron: an important factor in resuscitation?

BACKGROUND: Prooxidant activity of non-protein-bound iron (NPBI) is an important contributor to reactive oxygen species-induced injury after the resuscitation of critically ill patients. Plasma NPBI occurs in critically ill adults, children, and newborn babies, who often require resuscitation. The ability of the resuscitation fluids to bind iron and lower the patients' NPBI levels in vitro has not previously been studied. STUDY DESIGN AND METHODS: In an in vitro model, highly iron-saturated cord blood plasma from 10 preterm and 10 term babies was mixed with FFP, pasteurized plasma protein solution, and 0.9-percent saline. Plasma from 10 healthy adult volunteers was used as a control. Before and after the mixing with any resuscitation fluid, NPBI levels and ceruloplasmin iron-oxidizing and transferrin iron-binding antioxidant capacities were measured. RESULTS: After the in vitro mixing with FFP, the incidence and concentration of NPBI were markedly decreased and the iron-binding antioxidant capacity was increased in the plasma of the preterm and term babies. Being mixed with pasteurized plasma protein solution and 0.9-percent saline did not influence the iron-binding antioxidant capacity of newborn babies' plasma. In the control plasma, results were not changed after the mixing with any resuscitation fluid. In every group, the iron-oxidizing antioxidant capacity was not changed after the mixing with any fluid. CONCLUSION: Iron-induced oxidative tissue damage may be influenced by resuscitation fluids. In the ongoing debate over the choice of crystalloid or colloid resuscitation fluids, the influence of each fluid on the patient's antioxidant capacity warrants more attention.

Influence of inhibition of nitric oxide synthesis on cardiac function in the newborn lamb after hypoxic-ischemic injury.

The aim of the present study was to investigate the effect of immediate post-hypoxic-ischemic (HI) inhibition of nitric oxide synthesis by N(omega)-nitro-L-arginine (NLA) on cardiac function and reactive oxygen species production. Fifteen newborn lambs were subjected to severe HI. Upon resuscitation 5 received 10 mg NLA/kg, 4 40 mg NLA/kg and 6 a placebo. Left ventricular (LV) contractility, cardiac output (CO), non-protein-bound iron (NPBI), ratio of reduced/oxidized ascorbic acid, alpha-tocopherol, sulfhydryl groups and malondialdehyde were measured before and 15, 60 and 120 min after resuscitation. There was a significant decrease in CO in all 3 groups at 60 min post-HI (p < 0.05). Reactive oxygen species production was also highest at 60 min post-HI (significantly increased NPBI and decrease in sulfhydryl groups in control lambs; p < 0.05). These results suggest neither a positive nor a negative effect of nitric oxide synthesis inhibition on post-HI myocardial performance but may suggest a positive effect of NLA on reactive oxygen species-mediated post-HI damage.

Impaired plasma antioxidative defense and increased nontransferrin-bound iron during high-dose chemotherapy and radiochemotherapy preceding bone marrow transplantation.

To analyze the effects of radiochemotherapy on the pro-oxidative/antioxidative balance in plasma, we measured the total radical antioxidant parameter of plasma (TRAP) and single plasma antioxidants (uric acid, sulfhydryl groups, alpha-tocopherol, ubiquinone-10/total coenzyme-Q10 ratio, ascorbate, and bilirubin) every 12 h during high-dose chemotherapy and radiochemotherapy preceding bone marrow transplantation (BMT). Nontransferrin-bound iron (NTBI) was monitored as a potential pro-oxidant. Plasma levels of polyunsaturated fatty acids (PUFA) were measured as substrates, and thiobarbituric acid-reactive substances (TBARS) were measured as products of lipid peroxidation. Allantoin was analyzed as the product of uric acid oxidation. Patients receiving busulfan, VP-16, and cyclophosphamide (BU/VP/CY) (n = 8) were compared with those receiving total body irradiation in addition to VP-16 and cyclophosphamide (TBI/VP/CY) (n = 8). TRAP values were within the normal range before therapy and decreased after BU/VP/CY by 37% (p <. 02) and after TBI/VP/CY by 39% (p <.02). During TBI and after VP-16, a temporary increase in TRAP values occurred, which was not related to changes in individual antioxidants. In vitro experiments confirmed that VP-16 had an antioxidative effect. The concentration of uric acid declined in both groups and correlated with TRAP (BU/VP/CY: r =.80, p <.001; TBI/VP/CY: r =.84, p <.001). Levels of NTBI, which is normally not found in plasma, increased rapidly during conditioning therapy (p <.02 in both groups) and correlated inversely with TRAP (weighted intraindividual Spearman rank correlation coefficient for both groups: NTBI and TRAP: r = -.59, p <.001) and PUFA (in the radiochemotherapy group: r = -.67, p <.001). Whereas PUFA declined (p <.02 in both groups), TBARS increased (p <. 05 in both groups). Furthermore, an increase of allantoin and ubiquinone-10/total coenzyme-Q10 ratio in the BU/VP/CY group was found (allantoin: p <.02; ubiquinone-10/total coenzyme-Q10 ratio: p <.05). Antioxidants only partially recovered to baseline values until day 14 after BMT. Our findings indicate oxidative stress after high-dose radiochemotherapy and suggest a contribution of NTBI therein.

Plasma proteins in acute and chronic lung disease of the newborn.

This study compared plasma levels of albumin, transferrin, and ceruloplasmin in well preterm babies (n = 21) with those with respiratory distress syndrome (RDS, n = 13) and chronic lung disease (CLD, n = 13) over the first 28 postnatal days. Plasma lipid peroxidation, total radical trapping capacity (TRAP assay), and iron binding antioxidant capacity were also measured. In RDS and CLD albumin levels were decreased on days 1, 4 and 10; on day 10 albumin was lower in CLD compared to RDS (p < .05). After day 10 the levels were similar in all groups. The transferrin levels showed a similar trend. Ceruloplasmin levels did not differ, except for a higher day 28 level in CLD (p < .05). Albumin levels significantly decreased with increasing FiO2 and duration of oxygen therapy (within patient r = -0.30, p < .05 and r = -0.51, p < .005, respectively). On day 10, increasing oxygen therapy increased plasma lipid peroxidation (r = +0.49, p < .01), which was also significantly related to lower plasma protein levels (r = -0.42, p < .01). Lower plasma albumin and transferrin lowered the TRAP and iron binding antioxidant capacity, respectively (r = +0.36, p < .05, and r = +0.41, p < .005). Prediction of CLD using day 10 albumin levels had a specificity of 94%, but a sensitivity of only 50%. The interaction between oxygen toxicity and high ventilation pressures in immature babies appears to lower plasma proteins by increasing pulmonary permeability. The lower plasma albumin level was not useful in predicting the development of CLD; however, the fall in plasma transferrin and albumin will further decrease the preventive and chain-breaking antioxidant capacity of plasma of these ill babies.

Effect of allopurinol on postasphyxial free radical formation, cerebral hemodynamics, and electrical brain activity.

OBJECTIVE: Free radical-induced postasphyxial reperfusion injury has been recognized as an important cause of brain tissue damage. We investigated the effect of high-dose allopurinol (ALLO; 40 mg/kg), a xanthine-oxidase inhibitor and free radical scavenger, on free radical status in severely asphyxiated newborns and on postasphyxial cerebral perfusion and electrical brain activity. METHODS: Free radical status was assessed by serial plasma determination of nonprotein-bound iron (microM), antioxidative capacity, and malondialdehyde (MDA; microM). Cerebral perfusion was investigated by monitoring changes in cerebral blood volume (delta CBV; mL/100 g brain tissue) with near infrared spectroscopy; electrocortical brain activity (ECBA) was assessed in microvolts by cerebral function monitor. Eleven infants received 40 mg/kg ALLO intravenously, and 11 infants served as controls (CONT). Plasma nonprotein-bound iron, antioxidative capacity, and MDA were measured before 4 hours, between 16 and 20 hours, and at the second and third days of age. Changes in CBV and ECBA were monitored between 4 and 8, 16 and 20, 58 and 62, and 104 and 110 hours of age. RESULTS: Six CONT and two ALLO infants died after neurologic deterioration. No toxic side effects of ALLO were detected. Nonprotein-bound iron (mean +/- SEM) in the CONT group showed an initial rise (18.7 +/- 4.6 microM to 21.3 +/- 3.4 microM) but dropped to 7.4 +/- 3.5 microM at day 3; in the ALLO group it dropped from 15.5 +/- 4.6 microM to 0 microM at day 3. Uric acid was significantly lower in ALLO-treated infants from 16 hours of life on. MDA remained stable in the ALLO group, but increased in the CONT group at 8 to 16 hours versus < 4 hours (mean +/- SEM; 0.83 +/- 0.31 microM vs 0.50 +/- 0.14 microM). During 4 to 8 hours, delta CBV-CONT showed a larger drop than delta CBV-ALLO from baseline. During the subsequent registrations CBV remained stable in both groups. ECBA-CONT decreased, but ECBA-ALLO remained stable during 4 to 8 hours of age. Neonates who died had the largest drops in CBV and ECBA. CONCLUSION: This study suggests a beneficial effect of ALLO treatment on free radical formation, CBV, and electrical brain activity, without toxic side effects.

Oxidative stress during post-hypoxic-ischemic reperfusion in the newborn lamb: the effect of nitric oxide synthesis inhibition.

Post-hypoxic-ischemic (HI) reperfusion induces endothelium and neurons to produce excessive amounts of nitric oxide and superoxide, leading to peroxynitrite formation, release of protein-bound metal ions (i.e. iron), and cytotoxic oxidants. We produced severe HI in 18 newborn lambs and serially determined plasma prooxidants (non-protein-bound iron), lipid peroxidation (malondialdehyde), and antioxidative capacity [ratio of ascorbic acid/dehydroascorbic acid (AA/DHA), alpha-tocopherol, sulfhydryl groups, allantoin/uric acid ratio, and vitamin A] in blood effluent from the brain before and at 15, 60, 120, and 180 min after HI. The lambs were divided in three groups: six received a placebo (CONT), six received low dose (10 mg/kg/i.v.) N omega-nitro-L-arginine (NLA-10) to block nitric oxide production, and six received high dose NLA (40 mg/kg/i.v.; NLA-40), immediately after completion of HI. Non-protein-bound iron increased in all groups after HI but was significantly lower in both NLA groups at 180 min post-HI (p < 0.05), the AA/DHA ratio showed a consistent decrease in CONT (at 60 min post-HI, p < 0.05), but remained stable in NLA lambs. alpha-Tocopherol decreased steadily in the CONT, but not in the NLA lambs [180 post-H: 1.9 +/- 0.9 versus 4.2 +/- 0.7 microM (NLA-40), p < 0.05). Malondialdehyde was significantly higher in CONT lambs 120 min post-H compared with NLA groups [0.61 +/- 017 versus 0.44 +/- 0.05 microM (NLA-40), p < 0.05]. Vitamin A and sulfhydryl groups did not differ among groups. We conclude that post-H inhibition of nitric oxide synthesis diminishes non-protein-bound iron increment and preserves antioxidant capacity.

Uric acid and ascorbic acid redox ratios in plasma and tracheal aspirate of preterm babies with acute and chronic lung disease.

This study compared plasma redox ratios of uric acid and ascorbic acid in well preterm babies with those with respiratory distress syndrome (RDS) and chronic lung disease (CLD), and investigated the relationship between these ratios and their respective measurements in tracheal aspirate. On day 1 after birth, plasma allantoin and allantoin/uric acid ratio were elevated in CLD (p < .05), and both markers of oxidative stress enabled early prediction of development of CLD (sensitivity and specificity: 54 and 83%, respectively). The relation between allantoin production and oxidative stress is supported by the correlation between the allantoin level and oxygen therapy in both RDS and CLD (p < .05). Reduced and oxidize ascorbic acid in plasma decreased postnatally in all groups and their redox ratio remained stable. Uric acid and ascorbic acid redox ratios were significantly elevated in tracheal aspirates compared to plasma samples (p < .05), and there was a strong positive correlation between both ratios (p < .005). These markers may be useful in monitoring babies with respiratory distress.

Markers of oxidative stress and antioxidant activity in plasma and erythrocytes in neonatal respiratory distress syndrome.

Markers of oxidative stress and antioxidant activity in plasma and erythrocytes were studied for 14 d after birth in infants with neonatal respiratory distress syndrome (n = 9) and controls (n = 36). In plasma, the total radical trapping antioxidant capacity and the chain-breaking antioxidants vitamin C, sulfhydryl groups and bilirubin were similar. The differences in uric acid levels were not consistent, but vitamin E levels and vitamin E/total-lipid ratio were lower in the neonatal respiratory distress group (p < 0.01). In erythrocytes, the antioxidant enzymes glutathione peroxidase, glutathione reductase and superoxide dismutase did not differ postnatally. Indicators of oxidative damage in plasma (sulfhydryl/protein ratio and thiobarbituric acid reactive substances) showed the same postnatal course in both groups and were not influenced by oxygen therapy. In erythrocytes the reduced/oxidized glutathione ratio showed no consistent differences. In conclusion, this study, using erythrocytes and plasma, does not provide convincing evidence of oxidative damage and diminished antioxidant defenses in preterm infants with neonatal respiratory distress syndrome.

Red blood cell glutathione and plasma sulfhydryls in chronic lung disease of the newborn.

We compared the postnatal changes (days 1-28) in red blood cell glutathione and plasma sulfhydryl content in preterm babies developing chronic lung disease (CLD, n = 13) to those in babies with respiratory distress syndrome (RDS, n = 13) and control babies (n = 21). There were no initial differences in these measurements between the three groups. However, on day 28 in CLD and RDS the red blood cell glutathione was decreased compared to the control babies (p < 0.05). In CLD, there was a significant correlation between reduced/oxidized glutathione and (i) maximal FiO2 (r = -0.69, p < 0.05) and (ii) minimal a/A ratio (r = +0.73, p < 0.005). On day 28, although the plasma sulfhydryl level did not differ between the groups, the sulfhydryl/total protein ratio was decreased in CLD (p < 0.05). The late decrease in both glutathione and sulfhydryl/total protein ratio in babies with CLD suggests that oxidative stress is still ongoing at 28 days after birth and that the antioxidant capacity of their blood is still diminished at this time.

Influence of plasma preparations and donor red blood cells on the antioxidant capacity of blood from newborn babies: an in vitro study.

We investigated in an in vitro transfusion model the early effects of plasma preparations and donor red blood cells on the antioxidant capacity of the cord blood from babies. Addition of pasteurized plasma protein solution to plasma from babies decreased the peroxyl radical trapping capacity (p < 0.02). In contrast, fresh frozen plasma did not lower this capacity. Addition of adult donor red blood cells to the babies' red blood cells did not significantly decrease the glutathione-recycling capacity of the blood. On the basis of these in vitro results we hypothesize that the use of resuscitation fluids with low antioxidant capacity may temporarily decrease the ability of the baby to catabolize reactive oxygen species.

Inhalation of nitric oxide: effect on cerebral hemodynamics and activity, and antioxidant status in the newborn lamb.

Ventilation with nitric oxide (NO) is increasingly being used to treat pulmonary hypertension in the newborn. In the brain, NO has vasoactive properties and is involved in neurotransmission. However, the effect of inhaled NO on the cerebral blood flow (CBF) and on the cerebral activity is not known. Furthermore, there is little information on the influence of this free radical gas on the redox status in pulmonary vessels. We therefore investigated the effect of inhaled NO (2-60 ppm) on CBF, cerebral activity and redox status in blood effluent from the pulmonary circulation in 6 ventilated newborn lambs before and during group B streptococci (GBS)-induced pulmonary hypertension. Blood pressure in the pulmonary artery (P(ap)) and aorta (Pao), carotid artery blood flow (Qcar) to assess changes in CBF, and electrocortical activity were measured. Blood gases, indices of free radical status and methemoglobin were determined in blood samples obtained from the left ventricle. Inhalation of NO, before and during GBS-induced pulmonary hypertension, decreased P(ap) and PCO2 and increased PO2. Multiple linear regression revealed that Qcar was positively related to PCO2, but not to inhaled NO or PO2 before or during GBS conditions. Electrocortical activity and indices of antioxidative capacity and lipid peroxidation did not change significantly. Methemoglobin was not detected. In conclusion, inhalation of NO (up to 60 ppm) lowered P(ap) without directly affecting CBF, electrocortical activity, and redox status in the pulmonary vessels. CBF, however, can indirectly be influenced by NO-mediated changes in PCO2.

Lipid peroxidation in human milk and infant formula: effect of storage, tube feeding and exposure to phototherapy.

Preformed lipid peroxidation products present in the feed may contribute to the total reactive oxygen radical load infants have to deal with and may play a role in the pathogenesis of necrotizing enterocolitis and bronchopulmonary dysplasia. In this study, the occurrence of lipid peroxidation in human milk and feeding formulas for preterm babies was evaluated in vitro. Free linoleic acid (18:2) and its hydroperoxide (18:2OOH) were measured by gas chromatography-mass spectrometry and the concentration of 18:2OOH and the 18:2OOH/18:2 ratio were used as indices of peroxidation. In all feeds peroxidation products were present, but the proportion of peroxidized 18:2 was greater in infant formula. Storage of human milk (+4 degrees C for four days) increased lipid peroxidation. Exposure to light during tube feeding increased peroxidation in infant formula but not in human milk. Different procedures for preparation, storage and feeding may decrease the concentration of these potentially toxic peroxidized lipids in human milk and infant formula.

Induction of lipid peroxidation of pulmonary surfactant by plasma of preterm babies.

Respiratory distress syndrome of the preterm baby is believed to be caused by a deficiency of pulmonary surfactant and leakage of plasma into the alveolar spaces. Since the two pathogenetic factors seem to be inter-related, we postulated that peroxidation of surfactant by plasma iron could be the linking mechanism. We obtained cord blood samples from 22 preterm babies (mean gestational age 32.2 [SD 2.7] weeks) and 24 term babies (40.1 [1.6] weeks), and venous blood samples from 18 healthy adults. No adult had detectable non-protein-bound iron in the plasma, but 10/21 (48%) preterm babies and 6/24 (25%) term babies had detectable concentrations (rate difference 23% [95% Cl -5 to 51%], p = 0.20). Transferrin and haptoglobin concentrations were higher and free haemoglobin concentrations lower in adults than in babies (p < 0.005). Only transferrin differed significantly between term and preterm babies. Plasma from all 18 adults and from 23 (96%) term babies inhibited iron-catalysed lipid peroxidation of pulmonary surfactant liposomes. By contrast, plasma from 11 (50%) preterm babies stimulated such peroxidation (difference in stimulation rate 46% [20-71%], p < 0.005 for preterm vs term babies); the ability to stimulate peroxidation was related to the presence of non-protein-bound iron (p < 0.001). Peroxidation decreased in the babies when apotransferrin was added to plasma and in all subjects when alpha-tocopherol was incorporated into the surfactant liposomes. Lipid peroxidation of surfactant may contribute to the pathogenesis of respiratory distress syndrome. Possible therapeutic approaches are increasing babies' iron-binding capacity by plasma transfusions and increasing the antioxidant capacity of commercial surfactant.

Depletion of ATP but not of GSH affects viability of rat hepatocytes.

The purpose of this study was to examine the role of glutathione depletion and alterations in the energy status in the induction of acute cytotoxicity to freshly isolated rat hepatocytes. Depletion of intracellular glutathione by diethyl maleate and phorone to levels below 5% of control did not induce loss of viability nor loss of intracellular ATP. Ethacrynic acid, a compound known to deplete mitochondrial GSH in addition to cytosolic GSH, induced cell killing after a depletion of ATP, next to GSH depletion. The results confirmed that depletion of intracellular glutathione alone does not necessarily result in cell killing. Only when glutathione depletion is succeeded by reduction in ATP levels, loss of cell viability is observed. The relationship between alterations in the energy status and the induction of cell death was further substantiated by inhibition of glycolytic and mitochondrial ATP generation. Treatment of hepatocytes either with iodoacetic acid to inhibit glycolysis (in hepatocytes from fed rats) or with potassium cyanide to inhibit mitochondrial respiration (in hepatocytes from both fed and fasted rats) revealed that depletion of intracellular ATP could lead to lethal cell injury. The susceptibility of cells to metabolic inhibition was better reflected by the rate of reduction in the energy charge than by the reduction of ATP alone. In conclusion, our results suggest that alterations of the energy status may be a critical event in the induction of irreversible cell injury. Depletion of cellular GSH is only cytotoxic when followed by a reduction of the energy charge.

Recycling of glutathione during oxidative stress in erythrocytes of the newborn.

The ability of erythrocytes from newborn babies and adults to maintain reduced glutathione levels during oxidative stress was studied. In vitro incubation of erythrocytes with H2O2, with or without inactivation of catalase, caused a rapid depletion of reduced glutathione (GSH) and concomitant accumulation of oxidized glutathione followed by recovery of GSH and fall of oxidized glutathione to initial values in all subjects. Inactivation of catalase resulted in a 50% loss of intracellular glutathione (p less than 0.005), a larger maximum GSH depletion (p less than 0.05), and a longer GSH recovery time (p less than 0.005). Erythrocytes from newborn babies showed a smaller maximum GSH depletion (p less than 0.05) and a shorter GSH recovery time (p less than 0.005) compared with those from adults. These differences between the newborn and adult groups persisted after inactivation of catalase. An increase in maximum GSH depletion and GSH recovery time (p less than 0.005) was observed when a lower hematocrit was used for these GSH recovery studies. Effective glutathione recycling in erythrocytes may protect immature tissues of the newborn baby from peroxidative damage.