Fe2+/vitamin C — An appropriate in vitro model system to initiate lipid peroxidation

doi:10.1016/0162-0134(95)00032-1

Journal of Inorganic Biochemistry

Volume 61, Issue 1, January 1996, Pages 43-53

https://doi.org/10.1016/0162-0134(95)00032-1 Get rights and content

Abstract

The Fe²⁺/vitamin C system has often been used to initiate in vitro lipid peroxidation. In this paper, we attempt to locate the optimal conditions of the Fe²⁺/vitamin C system to be used in lipid peroxidation experiments. We have measured the quantity of lipid peroxidation through assessment of thiobarbituric acid reactive substances. We tested the influence of the Fe²⁺ and vitamin C concentration, the pH, the reaction temperature, and different buffer systems on the extent of lipid peroxidation in preparations of pig heart membranes. The optimum rate of lipid peroxidation occurred at an Fe²⁺/vitamin C ratio of 1:5. Decreasing pH and changing the buffer system resulted in changes of the extent of peroxidation. We also show that the EPR spin trap technique with 5,5-dimethyl-pyrroline-1-oxide (DMPO), which is used to detect free radicals in the Fe²⁺/vitamin C system, cannot work. This is because vitamin C immediately quenches the formed DMPO adducts by reduction. In addition, no evidence was found as to the nature of Fe²⁺/vitamin C formed radicals in experiments using superoxide dismutase, catalase or mannitol, butylhydroxyanisol, and α-tocopherole as radical scavengers.

References (36)

J.E. Schneider et al.
Free Rad. Biol. Med.
(1988)
G. Minotti et al.
Free Rad. Biol Med.
(1987)
N. Tamaru et al.
Prostaglandins, Leucotrienes and Essential Fatty Acids
(1993)
K. Kramer et al.
FEBS Lett.
(1986)
T. Kaneko et al.
Arch. Biochem. Biophys.
(1993)
E.R. Stadtman
Amer. J. Clin. Nutr.
(1991)
B. Halliwell
Free Rad. Res. Commun.
(1990)
R. Vetter et al.
FEBS Lett.
(1982)
O.H. Lowry et al.
J. Biol. Chem.
(1951)
D.M. Miller et al.
Arch. Biochem. Biophys.
(1989)

A. Scheschonka et al.

Chem.-Biol. Interactions

(1990)

J.M.C. Gutteridge et al.

Arch. Biochem. Biophys.

(1990)

B. Halliwell et al.

FEBS

(1992)

J.A. Buege et al.

Methods Enzymol.

(1978)

H. Verhagen et al.

Chem. Biol. Interactions

(1991)

J.M.C. Gutteridge

Free Rad. Res. Commun.

(1993)

I. Schimke et al.

Klin. Wochenschr.

(1986)

D. Harman

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The effect of antioxidants on the quality of cryopreserved semen in two salmonid fish, the brook trout (Salvelinus fontinalis) and the rainbow trout (Oncorhynchus mykiss)
2011, Theriogenology
Citation Excerpt :
The sperm lipid peroxidation test was performed with the thiobarbituric acid (TBA) reaction [17]. Lipid peroxidation was challenged with 0.04 mmol/l (final concentration) ferrous sulphate and 0.2 mmol/l (final concentration) ascorbic acid [18]. Then the sperm suspensions were incubated in a water bath at 37 °C for 3 h.
Until now the supplementation of cryopreservation extenders with antioxidants has not been examined in teleost fish. Therefore, the present study investigated whether addition of antioxidants (catalase, superoxide dismutase, peroxidase, reduced glutathione, reduced methione, mixtures of reduced and oxidized glutathione or methionine) to the cryopreservation extenders could increase the quality of frozen-thawed semen of brook trout, Salvelinus fontinalis, and rainbow trout, Oncorhynchus mykiss. In brook trout and rainbow trout semen post-thaw fertility and motility were evaluated and in brook trout additionally the membrane integrity, DNA integrity, and sperm lipid peroxidation were evaluated. The tested antioxidants affected the motility parameters, DNA integrity, and fertility of cryopreserved semen, but not the membrane integrity. Most of the observed effects were negative and only minor positive effects were found. In brook trout 1.5 mmol/l reduced methionine and a mixture of 1.5 mmol/l oxidized and reduced glutathione increased the swimming velocity of frozen-thawed semen. One hundred U/l catalase, 1.5 mmol/l reduced glutathione, and 1.5 mmol/l reduced methionine slightly, but not statistically significantly increased the semen post-thaw fertility. However, these effects were not detectable in rainbow trout. Antioxidative stress or damage seems to play no role during cryopreservation, as also in the lipid peroxidation test no differences were obtained between fresh and cryopreserved semen. Therefore, for routine cryopreservation extender supplementation with antioxidants is not recommended in brook trout and rainbow trout.
A comparative study on antioxidant systems in semen of species of the Percidae, Salmonidae, Cyprinidae, and Lotidae for improving semen storage techniques
2010, Aquaculture
Citation Excerpt :
The sperm lipid peroxidation test was performed with the thiobarbituric acid (TBA) reaction according to the method of Aitken et al. (1993). Ferrous sulphate (0.04 mmol/l, final concentration) and ascorbic acid (0.2 mmol/l, final concentration) was added to the sperm suspensions to challenge lipid peroxidation and then they were incubated in a water bath at 37 °C for 180 min (Haberland et al., 1996). Thereafter, 300 μl of TBA reaction mixture (1.0 ml of 7% sodium dodecyl sulphate, 10.0 ml of 0.1 mol/l HCl, 1.5 ml of 10% phosphotungstic acid, 5.0 ml of 0.67% TBA, and 0.5 ml of 0.2 mmol/l butylated hydroxytoluene) was added to 125 μl sperm suspension.
The present study investigated the antioxidant systems in semen of different teleost fish species (burbot — Lota lota, perch — Perca fluviatilis, bleak — Alburnus alburnus, brown trout — Salmo trutta) with the intention to define types and effective concentrations of antioxidants suitable for supplementation of sperm storage solutions and cryopreservation extenders.
Biochemical analysis revealed that in semen of L. lota, P. fluviatilis, A. alburnus, and S. trutta antioxidants (ascorbic acid, carnitine, glutathione, methionine, tocopherol, and uric acid) and oxidant defensive enzymes (catalase, gluthatione reductase, peroxidase, and superoxide dismutase) occurred in an almost similar qualitative and quantitative pattern whereby uric acid concentrations and superoxide dismutase activities were high while activities/concentrations of other enzymes and metabolites were low and/or fluctuating. Species-specific differences existed in the occurrence of catalase and carnitine.
Important antioxidants and oxidant defensive enzymes were tested on their sperm protective effect in in vitro experiments. Spermatozoa were incubated in sperm motility-inhibiting saline solutions containing the antioxidants or enzymes and thereafter motility was activated with distilled water and measured and membrane integrity and sperm lipid peroxidation were determined. The experiments demonstrated that uric acid is the major antioxidant of semen of the investigated species, as it improves the sperm motility and membrane integrity and decreases the sperm lipid peroxidation. Therefore, supplementation of sperm diluents with uric acid can be recommended to increase the quality of semen whereby the effective concentration was 0.5 mmol/l for all investigated species. Also methionine has importance as antioxidant in teleost fish semen whereby the oxidized form (methionine sulfoxide) was most effective to increase sperm motility and membrane integrity. The effective concentration was 1.5–3 mmol/l. Finally, catalase improved sperm motility and membrane integrity in all species with exception of A. alburnus and therefore it can be useful to protect spermatozoa from reactive oxygen species, too. The optimal activity was 2 kU/l for P. fluviatilis and L. lota, and 0.1 kU/l for S. trutta.
Antioxidant systems of brown trout (Salmo trutta f. fario) semen
2010, Animal Reproduction Science
The present study characterizes the antioxidant systems of brown trout, Salmo trutta, semen as supplementation of semen dilution media with antioxidants can be beneficial to improve techniques for semen storage and cryopreservation. Antioxidants and oxidant defensive enzymes of spermatozoa and seminal plasma were analyzed. To determine whether antioxidants and oxidant defensive enzymes have an effect on sperm functionality, in vitro experiments were performed. Selected antioxidants and oxidant defensive enzymes were added to sperm motility-inhibiting saline solution and their effects on sperm viability (motility when activated, membrane integrity, and lipid peroxidation) were measured.
In seminal plasma and spermatozoa the enzymes catalase, glutathione reductase, methionine sulfoxide reductase, peroxidase, and superoxide dismutase and the metabolites ascorbic acid, glutathione, methionine, tocopherol, and uric acid were detected. Of the enzymes superoxide dismutase had the highest activity, of the metabolites uric acid occurred in highest concentrations.
During in vitro incubation uric acid and catalase increased the sperm motility, sperm membrane integrity, and decreased the sperm lipid peroxidation in comparison to the control. However, catalase was effective only at an activity much higher than that occurring in seminal plasma. Reduced methionine increased the sperm motility and sperm membrane integrity and oxidized methionine the motility. However, neither reduced nor oxidized methionine decreased the sperm membrane lipid peroxidation. It is concluded, that uric acid is the main antioxidant of brown trout semen.
Antioxidant status of erythrocytes and their response to oxidative challenge in humans with argemone oil poisoning
2008, Toxicology and Applied Pharmacology
Oxidative damage of biomolecules and antioxidant status in erythrocytes of humans from an outbreak of argemone oil (AO) poisoning in Kannauj (India) and AO intoxicated experimental animals was investigated. Erythrocytes of the dropsy patients and AO treated rats were found to be more susceptible to 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) induced peroxidative stress. Significant decrease in RBC glutathione (GSH) levels (46, 63%) with concomitant enhancement in oxidized glutathione (172, 154%) levels was noticed in patients and AO intoxicated animals. Further, depletion of glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G-6-PDH) and glutathione-S-transferase (GST) (42–52%) was observed in dropsy patients. Oxidation of erythrocyte membrane lipids and proteins was increased (120–144%) in patients and AO treated animals (112–137%) along with 8-OHdG levels in whole blood (180%) of dropsy patients. A significant reduction in α-tocopherol content (68%) was noticed in erythrocytes of dropsy patients and hepatic, plasma and RBCs of AO treated rats (59–70%) thereby indicating the diminished antioxidant potential to scavenge free radicals or the limited transport of α-tocopherol from liver to RBCs leading to enhanced oxidation of lipids and proteins in erythrocytes. These studies implicate an important role of erythrocyte degradation in production of anemia and breathlessness in epidemic dropsy.
The effect of dietary supplementation with blueberry, α-tocopherol or astaxanthin on oxidative stability of Arctic char (Salvelinus alpinus) semen
2006, Theriogenology
The objective was to determine the oxidative stability of Arctic char (Salvelinus alpinus) semen following dietary supplementation with lowbush blueberry (Vaccinium angustifolium) product, α-tocopherol, α-tocopherol + blueberry product, or α-tocopherol + astaxanthin. Sperm lipid peroxidation was initiated by challenging with ferrous sulphate/ascorbic acid (Fe⁺⁺/Asc) at level of 0.04/0.2 mmol/L. Addition of blueberry, α-tocopherol, or both to char diets inhibited semen lipid peroxidation by: (a) decreasing the rate of sperm lipid peroxidation, an effect which was more pronounced with α-tocopherol treatments; and (b) increasing the antioxidant potential of seminal plasma, based on the lipid peroxidation process of sperm and an in vitro chicken brain tissue model. Dietary supplementation with astaxanthin and α-tocopherol had the same effect as the supplementation with α-tocopherol alone on inhibiting the lipid peroxidation process of sperm and chicken brain. Catalase-like activity increased significantly in sperm of fish fed α-tocopherol, blueberry, or both. There was a negative correlation (r = −0.397, P < 0.05) between catalase-like activity in sperm cells and the rate of sperm lipid peroxidation. Seminal plasma α-tocopherol levels increased significantly in fish supplemented with α-tocopherol alone or in combination with blueberry or astaxanthin. There were negative correlations between seminal plasma α-tocopherol levels and lipid peroxidation rates of sperm cells (r = −0.625, P < 0.01) and brain tissue (r = −0.606, P < 0.01). In conclusion, dietary supplementation of blueberry product or α-tocopherol inhibited lipid peroxidation in Arctic char semen. Further experiments are needed to test the effect of dietary blueberry and antioxidants on Arctic char semen quality during liquid and cryopreserved storage.
Suppression of steroidogenesis and activator protein-1 transcription factor activity in rat adrenals by vitamin e deficiency-induced chronic oxidative stress
2004, Journal of Nutritional Biochemistry
Excessive oxidative stress and associated macromolecular damage are considered to be key features of aging, and appear to contribute to the age-related decline in steroid hormone production in adrenal and testicular Leydig cells. The current studies were initiated to examine the potential mechanism by which excessive oxidative stress during aging attenuates the functional expression of the oxidant-responsive transcription factor Activator protein-1. Chronic oxidative stress was induced in vivo by maintaining groups of rats on a diet deficient in vitamin E for 6 months. Plasma, liver, and adrenal tissues from vitamin E–deficient animals had negligible levels of this vitamin and showed high susceptibility to in vitro lipid peroxidation. Synthesis and secretion of corticosterone in response to corticotropin (ACTH), dibutyryl-cAMP, or 20α-hydroxycholesterol in vitro was significantly reduced in adrenocortical cells prepared cells from rats deficient in vitamin E. AP-1 DNA-binding activity was diminished ∼55 % in adrenal extracts from vitamin E–deficient rats with no corresponding change in the binding activity of SP-1. The vitamin E deficiency–mediated loss of AP-1 activity was not due to an alteration in the dimeric composition of constituent proteins, but rather to a general down-regulation of steady-state levels of members of the Fos and Jun families of proteins. Interestingly, vitamin E deficiency also reduced the expression of the redox-regulated Ref-1 protein. Collectively these data demonstrate that chronic oxidative stress specifically down-regulates essential components of the AP-1 transcription factor complex, and suggest that aberrancies in AP-1 expression may adversely affect processes crucial for intracellular cholesterol transport and steroid hormone production.

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^☆: This work was supported by Bundesministerium für Forschung und Technologie der Bundesrepublik Deutschland, Förderschwerpunkt “Gestörte Herz- und Gefäfunktion” an der Medizinischen Fakultät der Humboldt-Universität zu Berlin, Charité, Projekt 3.5.

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Fe2+/vitamin C — An appropriate in vitro model system to initiate lipid peroxidation☆

Abstract

Free Rad. Biol. Med.

Free Rad. Biol Med.

Prostaglandins, Leucotrienes and Essential Fatty Acids

FEBS Lett.

Arch. Biochem. Biophys.

Amer. J. Clin. Nutr.

Free Rad. Res. Commun.

FEBS Lett.

J. Biol. Chem.

Arch. Biochem. Biophys.

Chem.-Biol. Interactions

Arch. Biochem. Biophys.

FEBS

Methods Enzymol.

Chem. Biol. Interactions

Free Rad. Res. Commun.

Klin. Wochenschr.

Fe²⁺/vitamin C — An appropriate in vitro model system to initiate lipid peroxidation☆