Thiobarbituric Acid-reactive Material Research Articles

Activities of Antioxidant Enzymes in Various Tissues of Male Fischer 344 Rats Are Altered by Food Restriction

The objective of this study was to determine how food restriction (40% restriction of food intake) altered the age-related changes in the activities of Cu,Zn superoxide dismutase, catalase and glutathione peroxidase in liver, brain cortex, heart, kidney and intestinal mucosa obtained from 6-, 16- and 26-mo-old male Fischer 344 rats. Food restriction increased the activity of one or more of the antioxidant enzymes in the liver, brain cortex, heart and kidney of the rats. However, the magnitude of the effect and the antioxidant enzyme(s) affected by food restriction varied from tissue to tissue, and food restriction had no significant effect on the activities of these enzymes in intestinal mucosa. Interestingly, the four tissues in which food restriction increased the activity of one or more of the antioxidant enzymes showed reduced lipid peroxidation as measured by thiobarbituric acid-reactive material. These data suggest that food restriction might enhance the survival of rodents by altering the levels of the antioxidant enzymes and hence reducing free radical damage.

Monitoring of morphological, biochemical and molecular responses of four contrasting barley genotypes under salinity stress

Salinity stress reduces crop growth and agricultural productions around the world. The effects of salinity stress on seed germination, some morphological, physiological and biochemistry traits, and expression of four genes at seedling stage in salt contrasting barley genotypes were investigated in the current study. Seed germination percentage and morphological traits were analyzed based on a factorial randomized complete design which ANOVA results showed that interaction between salinity (150 and 250 mM NaCl) and genotype (Afzal and Sahand (salt tolerant), and Fajr and Reyhan (sensitive)) was significat (p < 0.05). Also, physiological and biochemical traits were analyzed based on a factorial split plot based on randomized complete design which ANOVA results illustared that interaction between salinity, genotype and time (0, 12, 24 and 72 h after salt treatment) was significat (p < 0.05). Salinity stress significantly declined germination percentage, length and fresh weight of root and shoot, chlorophyll content while thiobarbituric acid reactive material ( TBARM), catalase activity, and proline values were increased in root and shoot of all genotypes at seedling stage. The aforementioned traits were more changed with increasing the salt concentration and the higher value of these traits except TBARM were observed in tolerant genotypes. Moreover, relative expression of four genes were investigated after 0, 12, 24 and 72 h of 150 and 250 mM NaCl treatments. The results revealed that expression of HvNHX3, HvCAT1, HvTIP2;3 and Hv3ARK were affected by salt stress and these genes play role in barley tolerance to mild and severe salinity stress based on their expression pattern.

Durum wheat seedlings in saline conditions: Salt spray versus root-zone salinity

Salinity is an increasingly serious problem with a strong negative impact on plant productivity. Though many studies have been made on salt stress induced by high NaCl concentrations in the root-zone, few data concern the response of plants to saline aerosol, one of the main constraints in coastal areas. In order to study more in depth wheat salinity tolerance and to evaluate damage and antioxidant response induced by various modes of salt application, seedlings of Triticum turgidum ssp. durum, cv. Cappelli were treated for 2 and 7 days with salt in the root-zone (0, 50 and 200 mM NaCl) or with salt spray (400 mM NaCl + 0 or 200 mM NaCl in the root-zone). Seedlings accumulated Na+ in their leaves and therefore part of their ability to tolerate high salinity seems to be due to Na+ leaf tissue tolerance. Durum wheat, confirmed as a partially tolerant plant, shows a higher damage under airborne salinity, when both an increase in TBA-reactive material (indicative of lipid peroxidation) and a decrease in root growth were recorded. A different antioxidant response was activated, depending on the type of salt supply. Salt treatment induced a depletion of the reducing power of both ascorbate and glutathione while the highest contents of proline were detected under salt spray conditions. In the short term catalase and ascorbate peroxidase co-operated with glutathione peroxidase in the scavenging of hydrogen peroxide, in particular in salt spray-treated plants. From our data, the durum wheat cultivar Cappelli seems to be sensitive to airborne salinity.

Differential response of parent and advanced mutant lines of wheat (Triticum aestivum L. cv. Tabasi) genotypes in antioxidant activity to salinity stress at seedling stage

Effect of sodium chloride salinity was studied in advanced mutant lines T-65-7-1 and T-67-60 (tolerant) and their wild type Tabasi (moderately tolerant) wheat genotypes under control and level of salinity (EC 6 ds m -1 ) at seedling stage. Salinity treatment decreased the contents of chlorophyll (CHL), carotenoids (CAR), α-Tocopherol (TOC) as well as enzyme activities including catalase (CAT), superoxide dismutase (SOD), ascorbic acid (AA), ascorbate peroxidase (APOX), and glutathione reductase (GR), whereas increased the contents of superoxide ion (O2 -), hydrogen peroxide (H2O2), thiobarbituric acid reactive material (TBARM) (measuring of lipid peroxidation) in wild type Tabasi cultivar. Salinity tolerant mutant line T-67-60 showed more enzymatic activities (CAT, SOD, ASA, APOX, GR) as well as CHL, CAR and TOC contents than T-65-7-1 mutant line and Tabasi cultivar. Interestingly, the contents of O2 -, H2O2 and TBARM in T-67-60 line showed less increased than T-65-7-1 line and Tabasi cultivar by salinity treatment. Base on these results the tolerance of T-67-60 mutant line to salinity, justified by intensified enzyme activities to control oxidative damage via quench ROS levels.

Radical scavenging activity of steviol glycosides, steviol glucuronide, hydroxytyrosol, metformin, aspirin and leaf extract of Stevia rebaudiana

Introduction: Reactive oxygen species (ROS), generated in many bioorganic redox processes, are the most dangerous by-products in the aerobic environment. The aim of this study was to compare the in vitro antioxidant activity of some chemical compounds and leaf extract of Stevia rebaudiana as natural antioxidant. Methods: The radical scavenging activity of ascorbic acid, quercetine, stevioside (ST), rebaudioside A (Reb A), steviol glucuronide (SVglu), glucose, sucrose, hydroxytyrosol, metformin and aspirin were measured and expressed as the inhibitory concentration in mM giving 50% reduction of radicals (IC50). Results: Ascorbic acid, quercetine, ST, Reb A, SVglu, glucose, sucrose, hydroxytyrosol, metformin and aspirin were active OH scavengers. Superoxide radicals were efficiently scavenged by ascorbic acid, SVglu, quercetine, hydroxytyrosol, ST, Reb A and aspirin. Quercetine, hydroxytyrosol, ascorbic acid and aspirin were active in limiting the amount of thiobarbituric acid (TBA) reactive material. Only ascorbic acid, quercetine and hydroxytyrosol showed DPPH and NO scavenging activity. Leaf extract of S. rebaudiana had an excellent ROS scavenging activity. Treatment of leaf extracts with PVPP and active charcoal removed a part of their scavenging activity. Results showed that sugars were not efficient ROS scavengers. Conclusion: Radical scavenging activity of steviol derivatives, aspirin, metformin and hydroxytyrosol might explain most of beneficial pharmacological effects on ROS related diseases, such as hypertension, type 2 diabetes, atherosclerosis, inflammation and certain forms of cancers. The results obtained in this study indicate that leaf extract of S. rebaudiana has a great potential to use as a natural antioxidant agent.

Biochemical Studies on Hemoglobin Modified with Reactive Oxygen Species (ROS)

Hemoglobin is the iron-containing oxygen transporting metalloprotein in the red cells of blood in mammals and other animals. Hemoprotein-mediated oxidative stress is thought to play a major role in pathophysiology of cerebral hemorrhage, blast pressure injury, crush injury, myocardial ischemia reperfusion injury. Hemoglobin undergoes oxidation-reduction reactions that lead to both generation and consumption of highly reactive oxygen and nitrogen species. In the present study, hemoglobin molecule was treated with hydrogen peroxide and the modification so incurred was analyzed by UV spectra, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and detection of carbonyl content. Our observations suggest that carbonyl content increases with increase in concentration of hydrogen peroxide. Production of hydroxyl radical was assessed by using benzoate degradation analysis. Our results was in tandem with the fact that hemoglobin on treatment with hydrogen peroxide rapidly generates free-radical species that can degrade benzoate to thiobarbituric acid reactive material which on reacting with thiobarbituric acid gives color. The increase in absorbance of ROS-modified hemoglobin at 532 nm shows the increase in benzoate degradation, which is a parameter of hydroxyl radical formation with increase in concentration of hydrogen peroxide. Modified hemoglobin was treated with catalase, mannitol, thiourea, glutathion, sodium benzoate and their effect were detected by spectroscopy and SDS-PAGE (12%). Substantial scavenging effect of aforementioned antioxidants reiterates the formation of hydroxyl radical. Catalase shows the maximum scavenging effect followed by thiourea and mannitol.

Measurement of myeloperoxidase and thiobarbituric acid-reactive material in plasma and bronchoalveolar lavage in E. coli-induced acute lung injury

Myeloperoxidase (MPO), which is exclusively contained in neutrophils, is released on their activation. Therefore, MPO may possibly be used as a parameter of neutrophil activation. Thiobarbituric acid-reactive material (TBARM) reflects lipid peroxidation and is a parameter of oxygen radical-mediated cell membrane damage. Using our guinea pig model of septic lung injury we measured MPO and TBARM in the setting of acute lung injury. The two experimental groups were saline controls (n = 8) and an E. coli septic group to which 2 x 10(9) live E. coli were administered intravenously (n = 8). Lung damage was assessed by measuring wet to dry lung weight ratio (W/D) and lung tissue to plasma accumulation of 125I-albumin (AL: albumin leakage). We measured MPO and TBARM in plasma and BAL fluid. Increased W/D and AL were observed in the E. coli group suggesting the development of acute lung injury. In the E. coli group, plasma MPO increased and MPO in BAL fluid was significantly increased as compared with the saline control group. There was no difference in plasma TBARM between the two groups, while TBARM in BAL fluid of the E. coli group was greater than in that of controls. Although BAL fluid TBARM correlated with both W/D and AL, there was no relation between BAL fluid MPO and either of these parameters. We conclude that TBARM in BAL fluid may be useful for assessing E. coli-induced acute lung injury in guinea pigs.

Effects of high-dose methylprednisolone on Na+-K+ ATPase and lipid peroxidation after experimental subarachnoid hemorrhage

The production of oxygen-free radicals and their subsequent peroxidative action on membrane unsaturated fatty acids could be enhanced after subarachnoid hemorrhage. High-dose methylprednisolone (30 mg/Kg i.v.) treatment can antagonize acute SAH-induced brain hypoperfusion and protect the ultrastructural integrity of endothelial cell membranes. Experimental subarachnoid hemorrhage (SAH) was induced in anesthesized rats by slow injection of 0.3 ml of autologous arterial blood into cisterna magna. Tissue lipid peroxidation, quantified as thiobarbituric acid reactive material (TBAR) and Na(+)-K+ ATPase activity were assayed in three different rat brain areas (cerebral cortex, hippocampus and brain stem) of controls (without any surgical manipulation), sham-operated (0.3 ml. of mock CSF into cisterna magna) and after SAH induction, at 1 h, 6 h and 48 h. Na(+)-K+ ATPase activity decreased in the cerebral cortex at 1 h and 6 h and in brain stem at 1 h after SAH, while the same enzymatic activity was unchanged in the hippocampus. High-dose methyl-prednisolone treatment (started immediately after SAH induction) enhanced the Na(+)-K+ ATPase activity until control levels. There was no significant difference in lipid peroxide content between sham-operated and hemorrhagic animals; however, the injection itself induces a transient increase of TBAR (1 h after injection) and methylprednisolone treatment decreases the products of lipid peroxidation in all brain areas.

Reduced extracellular phagocyte oxidative activity, antioxidant level changes and increased oxidative damage in healthy human blood as a function of age

Age-related changes in the blood antioxidant status, in the prooxidative activity of peripheral phagocytes and in the markers of oxidative injury were simultaneously examined in the circulation of 45 middle-aged and elderly healthy volunteers. The results showed a decrease in the opsonin-dependent and -independent extracellular-phagocyte oxidative activity, evaluated by means of luminol chemiluminescence. An increase in the portion of the mitochondrial superoxide generation within the total oxidative phagocyte response was evaluated by means of lucigenin chemiluminescence. The erythrocyte copper/zinc superoxide dismutase increased with age, while blood catalase and glutathione peroxidase activities remained unchanged. The levels of blood SH-groups decreased with age. An age-related increase in blood concentration of thiobarbituric acid-reactive material, a marker of oxidative damage, was detected. Some data, illustrating the existence of a delicate balance between oxidants and prooxidants, were also obtained. Further studies on the interrelationship between the components determining pro/antioxidative status in an organism may prove useful for developing a complex strategy in combating ageing.

Neuroprotective effects of infliximab in experimental spinal cord injury

Background The aim of the study is to assess the effects of infliximab, a TNF- α receptor blocker, in a spinal cord clip compression injury model. Methods Clip compression injury model was used for producing spinal cord injury on 32 adult, male Wistar rats (Gazi University Animal Research Laboratory, Ankara, Turkey). After exposing the vertebral column between T7 and T10, total laminectomy was performed with the assistance of a high-speed drill and a surgical microscope. The dura was left intact. Spinal cord injury was performed on all rats with application of a 70-g closing force aneurysm clip for 1 minute. The rats were randomly allocated into 4 groups. Control group received no further therapy, whereas the other 3 groups received methylprednisolone (30 mg/kg intraperitoneal), infliximab (5 mg/kg subcutaneous), and a mixture of these 2 agents. All rats were killed 72 hours later, and the level of lipid peroxides in traumatized spinal cord tissue were measured as thiobarbituric acid-reactive material and determined using the method of Mihara and Uchiyama (Determination of malonaldehyde precursor in tissue by thiobarbituric acid test. Anal Biochem 1978;86(1):271-8). Results Treatment with infliximab and methylprednisolone decreased MDA levels in rats with spinal cord injury with a statistically significant difference. In addition, combined therapy achieved a more profound decrease in tissue MDA levels, which was also statistically significant. Conclusions Infliximab is found as effective as methylprednisolone on spinal cord clip compression injury. Moreover, the combination of these 2 agents demonstrated higher efficacy suggesting a synergistic effect between these 2 agents. However, further studies regarding functional and behavioral analyses as well as biochemical markers are required.

The effect of mexiletine on the level of lipid peroxidation and apoptosis of endothelium following experimental subarachnoid hemorrhage

Objective: The role of apoptosis in etiopathogenesis of vasospasm is not clearly understood yet. It is widely accepted that protection of the endothelial cells from the process of apoptosis could have beneficial effects on cerebral vasospasm after subarachnoid hemorrhage (SAH). Mexiletine blocks sodium and calcium channels and activates ATP-sensitive K+ channels. Moreover, mexiletine is known to have potent antioxidant effects through inhibiting free-radical production.Methods: Twenty-one rabbits were allocated into three groups randomly. Group I was sham operated group (n=7). SAH occurred but no medication was given to the Group II rabbits (SAH only group) (n=7). Mexiletine (50 mg/kg, b.i.d., i.p.) was administered just before SAH and continued until 48 hours following SAH to the Group III rabbits (Mexiletine treated group) (n=7). The ApopTag peroxidase in situ apoptosis detection kit (Serologicals Corporation, former Intergen) was used to demonstrate apoptosis in a cross section of basillary arteries. Thiobarbituric acid reactive material was used to determine the lipid peroxidation levels.Results: There was a statistically significant difference between lipid peroxidation product levels of the control and SAH only groups (p<0.05). The level of lipid peroxidation production in Mexiletine treated group was significantly lower compared with SAH only group (p<0.05) but not significantly higher than the control group (p>0.05).Discussion: In the present study we investigated the antioxidant action of mexiletine on apoptosis of endothelium following a rabbit SAH model. This experimental study directly suggested that lipid peroxidation is an important step in development of apoptosis in endothelial cells and prevention of structural integrity of endothelial cell should play a beneficial role in attenuation of cerebral vasospasm. Mexiletine treatment prevented the increase in lipid peroxidation and cerebral vasospasm. Examination of endothelial cells by staining specific for apoptosis demonstrated significant protection of cell integrity in the treated group.

Lipid peroxidation of fish oils

Fish and fish oils are the richest sources of ω-3 fatty acids. However, they are susceptible to lipid peroxidation due to their high degree of unsaturation. In the present study, the level of thiobarbituric acid reactive material in various fish oils available in the market with and without added Vitamin E was determined. The peroxide levels in fish oil heated to food frying temperature of 180°C and the effect of addition of vitamin E has also been studied. The results indicate that the peroxide levels in almost all the products available in the market were abnormally high irrespective of their Vitamin E content. This might be due to the inefficient methods used for processing and storage of fish oils. Addition of vitamin E was found to have a significant effect in lowering the rate of peroxidation of fish oil during thermal stress, showing that association of antioxidants with ω-3 fatty acids lowers the rate of lipid peroxidation.

Chronic incidental lead ingestion in a group of captive-reared alligators ( Alligator mississippiensis): Possible contribution to reproductive failure

An American alligator ( Alligator mississippiensis) breeding facility using male and female alligators raised from artificially incubated eggs was established in 1975. These alligators first reproduced at 6 years of age as compared to 10–12 years in wild alligators, but the eggs produced showed a lower hatching rate than those collected from the wild. By age 21 reproduction had failed almost completely. The alligators were sacrificed and tissues collected at necropsy from 44 captive and 15 wild animals and assayed for metals. Results showed that captive alligators had significantly higher tissue levels of lead than wild alligators. Cadmium did not differ between wild and captive and selenium was 50% higher in wild than captive alligator kidneys. Bone lead in captive alligators was 252,443 ± 20,462 ng/g. High yolk lead was suggested as a probable cause for early embryonic death in alligator eggs. The high tissue lead levels in captive alligators was attributed to long-term consumption of nutria ( Myocastor coypus) meat contaminated with lead shot. Liver, ovary, and testis were assayed for lipid peroxidation using the thiobarbituric acid (TBA) test. Captive alligators had 3.6 fold increased TBA-reactive materials in the liver tissue compared to wild. Lipid peroxidation was strongly suspected as having been enhanced by consumption of rancid nutria meat containing lead.

Impairment of cell and plasma redox state in subjects professionally exposed to chromium.

Chromium (Cr) is widely used in chemical, tannery, building, and metal industries. More recently, it has been demonstrated that Cr induces oxidative stress in mouse brain. Nevertheless very few data exist on in vivo oxidative damage in humans exposed to Cr. Changes in antioxidant parameters both in plasma (acid ascorbic redox state and total antioxidant capacity) and in red blood cells (glutathione (GSH) redox state) of 40 subjects (age 37.65 +/- 7.46; M/F 20/20) professionally exposed to Cr who were recruited from metal, chemistry, and building industries were evaluated. We also evaluated the levels of lipoperoxidation (thiobarbituric acid-reactive material, TBA-RM) and thiol levels in plasma to assess the extent of oxidative stress state. To evaluate Cr exposure rate, we measured urinary-chromium (U-Cr) by an electrothermic atomization-atomic absorption spectrometry (ETA-AAS) method. In this study, we found that Cr exposure induced a decrease both in GSH (P < 0.0005) and GSH/oxidized glutathione (GSSG) ratio (P < 0.0001) in red blood cells from workers with respect to control subjects. Furthermore, we also demonstrated a significant decrease of plasma acid ascorbic levels (45.7 +/- 14.9 vs. 53.5 +/- 16.5 micromol/L; P < 0.05) and in total plasma antioxidant capacity (1,126.3 +/- 212.2 vs. 1,266.9 +/- 207.8 micromol/L; P < 0.05) in subjects exposed to Cr. No difference was found with regard to TBA-RM and thiol levels. This study demonstrated that in humans, an oxidative stress occurs for Cr exposures as low as those considered safe. This oxidative stress appears to be able to affect intracellular and plasmatic antioxidant defense.

Dynamics of free radical processes in acute ischemic stroke: influence on neurological status and outcome.

The dynamics of free radical processes during the acute stage of ischemic stroke and their relationship with the clinical status of patients were studied. An enhanced extracellular generation of reactive oxygen species (ROS) by peripheral phagocytes was observed in severe stroke patients during the whole acute stage. This generation correlated positively with the size of infarct, the severity of neurological deficit and handicap and correlated negatively with the improvement of the neurological status of patients. An increase in the activity of two enzymes from the antioxidant defense mechanism, catalase and glutathione peroxidase, was registered during the whole acute phase of stroke, regardless of its severity. The concentration of lipid peroxidation products increased over time. Blood concentration of thiobarbituric acid-reactive material (TBARM) correlated positively with the size of infarct, the severity of neurological deficit and handicap. In conclusion, extracellular ROS generation by phagocytes and blood TBARM concentration could be used as indicators for stroke outcome.

Diabetic ketoacidosis induces in vivo activation of human T-lymphocytes

Diabetic ketoacidosis (DKA) is an inflammatory state associated with immune responses in polymorphonuclear cells (PMN). Activation of subgroup of T-lymphocytes in PMN of DKA patients, however, is not known. We studied in vivo activation of CD4 and CD8 lymphocytes by measuring de novo growth factor receptor for insulin, IGF-1, and IL-2 in eight patients on admission and at resolution of DKA, and compared them with matched controls. The presence of these receptors was demonstrated in all patients’ lymphocytes on admission, but not in control subjects. This event was associated with increased levels of thiobarbituric acid-reacting material and dichlorofluorescien, as markers of oxidative stress. Based on these new findings and works in the literature, we hypothesize that hyperglycemia/ketosis results in increased reactive oxygen species, leading to increased levels of cytokines and emergence of growth factor receptors. We propose DKA changes the T-lymphocytes to insulin sensitive tissues as a compensatory mechanism.

Oxidative stress in the chronic phase after stroke

The spontaneous and the stimulated extracellular generation of reactive oxygen species (ROS) by peripheral phagocytes, the blood antioxidant capacity and the degree of oxidative damage were evaluated in patients with severe ischemic and hemorrhagic stroke in the chronic phase of disease. It was found in patients compared to the control group that: (i) the spontaneous phagocyte oxidative activity was enhanced independently of the type of stroke and the time elapsed after stroke onset; (ii) there was no difference in the extracellular ROS generation stimulated by opsonin-dependent and independent receptor mechanisms; (iii) there was no change in the indices of blood antioxidant capacity; (iv) the concentration of plasma lipid peroxides was enhanced regardless of the type of stroke, but it significantly increased over time; and (v) the concentration of blood thiobarbituric acid-reactive material was also enhanced. It was independent of the type of stroke and remained elevated during the whole period studied. We have demonstrated an enhanced spontaneous phagocyte oxidative activity and oxidative damage to lipids in patients in the chronic phase after stroke. The elimination of generated ROS and products of lipid peroxidation from the circulation could prevent the aggravation of chronic vascular injury in patients and could reduce the possibility of a subsequent stroke. This suggests the need for complex therapy, including antioxidant treatment directed to exclude the effects of free radicals, after the oxidative stress of stroke.

Differences in oxidative stress in heavy metal resistant and sensitive clones of Salix viminalis

Many stress mechanisms, such as antioxidative mechanisms, are known to be activated by heavy metals. The aim of this work was to investigate whether oxidative stress and related mechanisms were expressed differently in plants with different resistances to heavy metals. Different clones of Salix viminalis with different resistances to Cd, Cu, and Zn were cultivated hydroponically in the presence of 7 μmol/L Cd, 3 μmol/L Cu or 70 μmol/L Zn for 20 days. The clones were then compared with regard to the concentrations of free radicals, estimated by measuring thiobarbituric acid-reactive material (TBA-rm) and glutathione (GSH). The activity of the enzymes, aspartate peroxidase (APX), guaiacol peroxidase (GPX), superoxide dismutase (SOD), and catalase (CAT) was also analysed. Salicylic acid was also measured since it is known to be involved in antioxidative activities. The results showed that some differences could be observed between resistant and sensitive clones. The SOD activity was higher in untreated resistant clones compared with the sensitive ones. Under metal treatment, however, the SOD activity was similar. Furthermore, TBA-rm was higher in shoots of resistant clones compared to sensitive ones, while the opposite was found in roots.

Progressively motile human spermatozoa are well protected against in vitro lipid peroxidation imposed by induced oxidative stress.

Semen samples of 24 patients were analysed. Volumes were measured and the numbers of progressively motile (PMS), motile (MS) and nonmotile spermatozoa (NMS) were determined. These 24 samples appeared to show a large variation in motility percentages and numbers. Spermatozoa of these semen samples were isolated from the seminal plasma and exposed to induced radical oxygen stress imposed by iron/ascorbate. Lipid peroxidation (LPO) was quantified as thiobarbituric acid reactive material. The contributions of PMS, MS and NMS were also estimated. It was found that the PMS did not contribute to the formation of lipid peroxides. The cellular radical defence system of PMS may offer them adequate protection against the harsh conditions of radical oxygen stress. Stepwise regression analyses showed that only the population of NMS contributed significantly to the explanation of the variance in LPO production (R2 = 0.56, P < 0.001). Pre-existing membrane lipid peroxides were not detected in spermatozoa. It is therefore suggested that LPO takes place only after radical oxygen stress has exhausted the cellular defence system. LPO is not the initial, but one of the later, events leading to the death of spermatozoa. It is concluded that the population of progressively motile spermatozoa in semen samples does not contribute to the production of thiobarbituric acid reactive substances as induced by in vitro radical oxygen stress.

Dependence of lipid peroxidation on pigmentation of the porcine iris

Melanin has been shown to act as antioxidant in lipid peroxidation studies. We have now investigated lipid peroxidation in dependence on stromal pigmentation in isolated porcine irises. The same number of lightly pigmented and heavily pigmented porcine irises (visual selection) were homogenized in buffer (50 mmol/l Na2HPO4, 50 mmol/l NaH2PO4 and 4 mmol/l sodium azide; 1:20 w/v). 500 microliters homogenate were incubated at 37 degrees C for 5, 10, 20 and 40 min in absence and presence of Fe2+ as inducer of lipid peroxidation. Lipid peroxidation was assayed by the thiobarbituric acid (TBA) test. Results are expressed as nmol of TBA reactive material produced (TBAR) per mg protein. Fe2+ concentration of the supernatant was determined spectrophotometrically with phenanthroline. 70 mumol/l, 180 mumol/l and 360 mumol/l Fe2+ induced lipid peroxidation. A plateau region was reached after 20 min. Lipid peroxidation differed in dependence on stromal pigmentation in porcine irises by a factor of 2.8. 180 mumol/l Fe2+ induced 1.373 +/- 0.138 nmol TBAR/mg protein in lightly pigmented irises compared to 0.491 +/- 0.125 nmol TBAR/mg protein in heavily pigmented irises after 10 min incubation (p < 0.0001, n = 4). On the other hand, the content of Fe2+ in the supernatant was the same within error. There was a stronger induction of lipid peroxidation in lightly pigmented porcine irises compared to heavily pigmented porcine irises. This effect may be related to the difference in stromal melanin content and its antioxidant activity.