Snežana Spasić

Bench-to-bedside review: Sepsis - from the redox point of view

The pathogenesis of sepsis and its progression to multiple organ dysfunction syndrome and septic shock have been the subject of investigations for nearly half a century. Controversies still exist with regard to understanding the molecular pathophysiology of sepsis in relation to the complex roles played by reactive oxygen species, nitric oxide, complements and cytokines. In the present review we categorise the key turning points in sepsis development and outline the most probable sequence of events leading to cellular dysfunction and organ failure under septic conditions. We have applied an integrative approach in order to fuse current state-of-the-art knowledge about redox processes involving hydrogen peroxide, nitric oxide, superoxide, peroxynitrite and hydroxyl radical, which lead to mitochondrial respiratory dysfunction. Finally, from this point of view, the potential of redox therapy targeting sepsis is discussed.

Relevance of the capacity of phosphorylated fructose to scavenge the hydroxyl radical

The hydroxyl radical (*OH) has detrimental biological activity due to its very high reactivity. Our experiments were designed to determine the effects of equimolar concentrations of glucose, fructose and mannitol and three phosphorylated forms of fructose (fructose-1-phosphate (F1P); fructose-6-phosphate (F6P); and fructose-1,6-bis(phosphate) (F16BP)) on *OH radical production via the Fenton reaction. EPR spectroscopy using spin-trap DEPMPO was applied to detect radical production. We found that the percentage inhibition of *OH radical formation decreased in the order F16BP>F1P>F6P>fructose>mannitol=glucose. As ketoses can sequester redox-active iron thus preventing the Fenton reaction, the Haber-Weiss-like system was also employed to generate *OH, so that the effect of iron sequestration could be distinguished from direct *OH radical scavenging. In the latter system, the rank order of *OH scavenging activity was F16BP>F1P>F6P>fructose=mannitol=glucose. Our results clearly demonstrate that intracellular phosphorylated forms of fructose have more scavenging properties than fructose or glucose, leading us to conclude that the acute administration of fructose could overcome the bodys reaction to exogenous antioxidants during appropriate therapy in certain pathophysiological conditions related to oxidative stress, such as sepsis, neurodegenerative diseases, atherosclerosis, malignancy, and some complications of pregnancy.

Bioavailability and catalytic properties of copper and iron for Fenton chemistry in human cerebrospinal fluid

Abstract A breakdown in homeostasis of redox-active metals represents an important factor for neurodegeneration. We have used EPR spectroscopy and BMPO spin-trap to investigate the catalytic properties and ligand modulation of redox activity of copper and iron in human cerebrospinal fluid (CSF). In contrast to iron, copper supplementation provoked a statistically significant increase in hydroxyl free radical generation in CSF treated with H2O2. However, in a binary copper/iron containing Fenton system, iron catalytically activated copper. The chelator EDTA, which represents a model of physiological metal ligands, completely prevented coppers redox activity in CSF, while iron chelation led to a significant increase in hydroxyl radical generation, indicating that copper and iron do not only have diverse catalytic properties in the CSF but also that their redox activities are differently modulated by ligands. The application of DDC reduced hydroxyl radical generation in the CSF containing catalytically active metals (free Cu2+ or Fe3+-EDTA complex). We conclude that chelators, such as DDC, are capable of preventing the prooxidative activity of both metals and may be suitable for reducing hydroxyl radical formation in certain pathophysiological settings.

Differences in direct pharmacologic effects and antioxidative properties of mature breast milk and infant formulas

OBJECTIVE Early-onset and exclusive breast-feeding provides a significant health benefit to infants compared with infant formulas. The aim of this study was to compare mature breast milk with standard infant formulas by examining their effects on non-vascular smooth muscle contraction and their antioxidative properties. METHODS The pharmacologic effects of breast milk and formulas were examined using a model system of the rat uterine smooth muscle contraction. Electron paramagnetic resonance spin-trapping spectroscopy was used to compare the antioxidative capacities of breast milk (obtained in the ninth week of lactation) with commercial infant formulas against hydroxyl radical production in the Fenton reaction. The activities of superoxide dismutase, glutathione peroxidase, and the sulfhydryl group were determined in the breast milk and infant formulas. RESULTS In contrast to the infant formulas, breast milk exerted a relaxing effect on isolated non-vascular smooth muscle. In general, breast milk showed higher antioxidative activity compared with the infant formulas. In all samples, the generation of hydroxyl radicals led to the formation of carbon-centered and ascorbyl radicals. CONCLUSIONS Human milk exerts direct pharmacologic relaxation effects and provides better antioxidant protection compared with infant formulas because of the presence of specific enzymatic components, such as human superoxide dismutase. We propose that these effects should be advantageous to an infants gastrointestinal tract by supporting the normal work of the smooth musculature and maintaining redox homeostasis and may represent one of the mechanisms by which breast-feeding benefits health.

Antioxidative Activity of Colostrum and Human Milk: Effects of Pasteurization and Storage

Objectives: Milk banks collect, pasteurize, and freeze/store human milk. The processing may alter redox properties of milk, but the effects have not been fully examined. Methods: We collected 10 mature milk and 10 colostrum samples and applied a battery of biochemical assays and electron paramagnetic resonance spectroscopy to inspect changes that milk undergoes with pasteurization and 30 days storage at −20°C. Results: Pasteurization and storage of raw milk did not affect total nonenzymatic antioxidative capacity, but specific components and features were altered. Urate radical and ascorbyl radical emerge as products of exposure of milk to hydroxyl radical-generating system. Processing shifted the load of antioxidative activity from ascorbate to urate and lowered the capacity of milk to diminish hydroxyl radical. Pasteurization caused a significant drop in the activity of 2 major antioxidative enzymes—superoxide dismutase and glutathione peroxidase, whereas freezing/storage of raw milk affected only superoxide dismutase. Colostrum showed drastically higher total nonenzymatic antioxidative capacity, hydroxyl radical scavenging ability, and glutathione reductase activity compared with mature milk. Conclusions: Pasteurization and storage affect nonenzymatic and enzymatic antioxidative agents in human milk. It appears that nonenzymatic antioxidative systems in colostrum and milk are different. The effects of processing may be partially compensated by fortification/spiking with ascorbate before use.

Redox therapy in neonatal sepsis: reasons, targets, strategy, and agents.

ABSTRACT Neonatal sepsis is one of the most fulminating conditions in neonatal intensive care units. Antipathogen and supportive care are administered routinely, but do not deliver satisfactory results. In addition, the efforts to treat neonatal sepsis with anti-inflammatory agents have generally shown to be futile. The accumulating data imply that intracellular redox changes intertwined into neonatal sepsis redox cycle represent the main cause of dysfunction of mitochondria and cells in neonatal sepsis. Our aim here is to support the new philosophy in neonatal sepsis treatment, which involves the integration of mechanisms that are responsible for cellular dysfunction and organ failure, the recognition of the most important targets, and the selection of safe agents that can stop the neonatal sepsis redox cycle by hitting the hot spots. Redox-active agents that could be beneficial for neonatal sepsis treatment according to these criteria include lactoferrin, interleukin 10, zinc and selenium supplements, ibuprofen, edaravone, and pentoxifylline.

Systemic NF-kappaB activation in blood cells of breast cancer patients.

There is a well-established role for reactive oxygen and nitrogen species, chronic inflammation and immune response in the pathogenesis of breast cancer. Complex interactions between breast cancer cells and surrounding blood vessels are prerequisites for cancer growth and invasion. Reports in the literature concerning the systemic response to, and the effect of, common breast cancer therapy on NF-kappaB and antioxidative defence enzyme expression and activity under clinical conditions are scarce. We determined these parameters in whole blood cell lysate from 16 women with breast cancer before and after combined (cyclophosphamide, doxorubicin, 5-fluorouracil; CAF) therapy and compared the results with 16 healthy women. Significantly higher levels of NF-kappaB and Mn-SOD (both their protein level and their activity) were found in breast cancer patients before and after CAF therapy, in comparison with healthy women. In parallel measurements, no change in the level or activity of catalase (CAT) was detected. According to our findings, it appears that breast cancer creates conditions that increase the level of hydrogen peroxide in the circulating cells and that the applied CAF therapy fails to compensate, therefore creating systemic conditions that favour survival and invasion of breast cancer cells.

Metals and electrolytes in sclerotic hippocampi in patients with drug-resistant mesial temporal lobe epilepsy

An altered metal and electrolyte profile has been implicated in the pathologic mechanisms of chronic epilepsy; however, no study has comprehensively measured hippocampal concentrations of these elements in patients with mesial temporal lobe epilepsy and hippocampal sclerosis (mTLE‐HS). We therefore analyzed hippocampi of 24 patients with drug‐resistant mTLE‐HS (mean age 35.6 ± 9.4 years) who underwent anterior temporal lobe resection and amygdalohippocampectomy and 17 hippocampi obtained by autopsy from 13 controls (mean age 40.5 ± 12.9 years), using inductively coupled plasma optical emission spectrometry (ICP‐OES). Epileptic hippocampi showed significantly lower concentrations (μg/g of tissue) of copper (HS: 2.34 ± 0.12; control [C]: 3.57 ± 0.33; p < 0.001), manganese (HS: 0.205 ± 0.030; C: 0.409 ± 0.064; p = 0.004), and potassium (HS: 2,001 ± 59; C: 2,322 ± 61; p < 0.001), and increased sodium levels (HS: 1,131 ± 22; C: 1,040 ± 25; p = 0.010). Zinc, iron, calcium, and magnesium levels did not differ in HS and controls. In summary, copper and manganese levels are deficient, whereas iron level is unchanged in hippocampi from patients with mTLE‐HS. Our results provide a basis for understanding the potential involvement of different metals and electrolytes in the pathology of HS.

Antioxidative system in the erythrocytes of preterm neonates with sepsis: the effects of vitamin E supplementation

Background Vitamin E is routinely supplemented to preterm babies, including those with neonatal sepsis. Our aim was to examine the effects of neonatal sepsis and vitamin E on antioxidative system (AOS) in the blood. Methods A prospective, randomized, open label study involved 65 preterm neonates (control/sepsis – 34/31), which were divided into two subgroups – non-supplemented and supplemented with vitamin E (25 IU/day for 60 days). The activities of superoxide dismutase, catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) were determined in erythrocytes at days 0, 30, and 60, following sepsis diagnosis. Results There was no difference in the activity of AOS between controls and neonates with ongoing sepsis. At 60 days, septic neonates showed higher CAT activity compared to controls (P = 0.027), and lower GPx activity compared to 0 days (P = 0.022). The later was mitigated by vitamin E, which on the other hand provoked lower GPx activity at 30 days, compared to untreated septic neonates (P = 0.014). In addition, vitamin E suppressed GR activity in septic neonates (P = 0.025 and P = 0.017 at 30 and 60 days). Finally, vitamin E supplementation in control neonates provoked a significant increase of GPx activity (P = 0.015 at 60 days). Conclusions The absence of altered redox settings in the blood of neonates during sepsis episode, and vitamin E-provoked decrease in the activity of some components of AOS, suggest that the supplementation of vitamin E in these patients might not be rational.

Electrochemical monitoring of the breast milk quality

The electrochemical techniques were used to determine the total antioxidant capacity of breast milks and the results were compared with a commonly used spectrophotometric (2,2-diphenyl-1-picrylhydrazyl, DPPH) method. Breast milk from mothers of preterm infants was monitored in three lactation phases and after storage of expressed milk by monitoring changes in the total antioxidant capacity over a two year period. Statistical analysis showed there was no significant difference between the ability of the three methods to detect changes in breast milk after storage. Either of the electrochemical techniques studied could be successfully used to replace the time-consuming spectrophotometric method and can be applied to clinical trials.