Reinterpreting the best biomarker of oxidative stress: The 8-iso-prostaglandin F2α/prostaglandin F2α ratio shows complex origins of lipid peroxidation biomarkers in animal models

Abstract

Oxidative stress is elevated in numerous environmental exposures and diseases. Millions of dollars have been spent to try to ameliorate this damaging process using anti-oxidant therapies. Currently, the best accepted biomarker of oxidative stress is the lipid oxidation product 8-iso-prostaglandin F2α (8-iso-PGF2α), which has been measured in over a thousand human and animal studies. 8-iso-PGF2α generation has been exclusively attributed to nonenzymatic chemical lipid peroxidation (CLP). However, 8-iso-PGF2α can also be produced enzymatically by prostaglandin-endoperoxide synthases (PGHS) in vivo. When failing to account for PGHS-dependent generation, 8-iso-PGF2α cannot be interpreted as a selective biomarker of oxidative stress. We investigated the formation of 8-iso-PGF2α in rats exposed to carbon tetrachloride (CCl4) or lipopolysaccharide (LPS) using the 8-iso-PGF2α/PGF2α ratio to quantitatively determine the source(s) of 8-iso-PGF2α. Upon exposure to a 120mg/kg dose of CCl4, the contribution of CLP accounted for only 55.6±19.4% of measured 8-iso-PGF2α, whereas in the 1200mg/kg dose, CLP was the predominant source of 8-iso-PGF2α (86.6±8.0% of total). In contrast to CCl4, exposure to 0.5mg/kg LPS was characterized by a significant increase in both the contribution of PGHS (59.5±7.0) and CLP (40.5±14.0%). In conclusion, significant generation of 8-iso-PGF2α occurs through enzymatic as well as chemical lipid peroxidation. The distribution of the contribution is dependent on the exposure agent as well as the dose. The 8-iso-PGF2α/PGF2α ratio accurately determines the source of 8-iso-PGF2α and provides an absolute measure of oxidative stress in vivo.

Keywords: 8-iso-PGF(2α)/PGF(2α) ratio; Biomarkers; F(2)-isoprostanes; Inflammation; Lipid peroxidation; Oxidative stress.

Figure 1:. Exposure to carbon tetrachloride increases the levels of 8-iso-PGF_2α generated mainly by chemical lipid peroxidation (CLP) in non-saponified plasma.

A: Exposure to carbon tetrachloride induces a statistically significant increase in free 8-iso-PGF_2α. A small increase in PGF_2α is seen concomitantly. This increase in PGF_2α differs significantly from control only at the 1200 mg/kg dose. B: The 8-iso-PGF_2α / PGF_2α ratio increases depending on the carbon tetrachloride dose. C: The sources of the total measured 8-iso-PGF_2α levels distinguished using the calculated 8-iso-PGF_2α / PGF_2α ratio and equation 1. A dose-dependent increase in 8-iso-PGF_2α from CLP is observed upon exposure with carbon tetrachloride with a significant but non-dose-dependent increase in the contribution of PGHS. D: Rats are pretreated with indomethacin, which inhibited PGHS activity significantly as seen in the decrease in PGF_2α levels. Increases in 8-iso-PGF_2α are observed similar to the non-pretreated animals upon carbon tetrachloride exposure. E: The 8-iso-PGF_2α / PGF_2α ratio does not change with increasing carbon tetrachloride dose after pretreatment with indomethacin. F: After distinguishing the sources of 8-iso-PGF_2α, the increase in the contribution of 8-iso-PGF_2α from PGHS as well as CLP is significantly decreased from indomethacin-pretreated animals; however, the increase in CLP persists even with indomethacin pretreatment. No increase in the contribution of PGHS compared to control is observed after indomethacin pretreatment. Six animals were treated per group. * Different from control (p<0.05), ** different from control and other dose (p<0.05), § different from non-pretreated group (p<0.05), # different from other dose but not control (p<0.05). Bars display mean ± SE.

Figure 2:. Carbon tetrachloride induces a time-dependent increase in 8-iso-PGF_2α generated predominantly by chemical lipid peroxidation in non-saponified plasma.

A: A time-dependent increase in 8-iso-PGF_2α and PGF_2α is observed upon exposure to 1200 mg/kg carbon tetrachloride; however, there is basal formation of ~1 ng/mL PGF_2α which causes an offset between the two curves due to continued PGF_2α formation by PGHS. B: The 8-iso-PGF_2α / PGF_2α ratio increases over control at the 2-hour time point and remains increased at the 7-hour time point. C: The sources of the 8-iso-PGF_2α levels from Figure 2A are distinguished using the calculated 8-iso-PGF_2α / PGF_2α ratio and equation 1. A time-dependent increase in the contribution of CLP is seen upon exposure. A significant increase in the contribution of PGHS is also observed; however, this does not change with time. D: After pretreatment with indomethacin, a time-dependent increase in 8-iso-PGF_2α and PGF_2α is still observed upon exposure to 1200 mg/kg carbon tetrachloride; both 8-iso-PGF_2α and PGF_2α are produced at nearly equal levels at each time point investigated. E: The 8-iso-PGF_2α / PGF_2α ratio remains near 1 throughout the time course after pretreatment with indomethacin before carbon tetrachloride exposure. F: After pretreatment with indomethacin, the increase in the contribution of PGHS is significantly inhibited. The time-dependent increase in CLP is different for non pretreated rats only at the 7-hour time point. Six animals were treated per time point. * Different from control (p<0.05), ** different from control and other time point (p<0.05), § different from non-pretreated group (p<0.05), # different from other dose but not control (p<0.05). Points display mean ± SE.

Figure 3:. Exposure to LPS induces the contribution of PGHS to the generation of free 8-iso-PGF_2α in non-saponified plasma.

A: After exposure to LPS, the levels of 8-iso-PGF_2α significantly increase compared to control at both doses, but PGF_2α significantly increases only at the high dose. B: The 8-iso-PGF_2α / PGF_2α ratio increases only slightly upon exposure to LPS at both the 0.5 mg/kg and 1.5 mg/kg dose. C: When the sources of 8-iso-PGF_2α are distinguished, a significant contribution in both LPS doses is seen for PGHS as well as CLP at the 0.5 mg/kg dose. Only the contribution of PGHS is increased significantly at the 1.5 mg/kg dose. D: After pretreatment with indomethacin, both 8-iso-PGF_2α and PGF_2α are significantly inhibited. E: The 8-iso-PGF_2α / PGF_2α ratio does not change from control in both LPS doses. F: The contributions of both PGHS and CLP to the 8-iso-PGF_2α levels are significantly inhibited with indomethacin pretreatment. Six animals were treated per group. * Different from control (p<0.05), different from non-pretreated group (p<0.05), § different from non-pretreated group (p<0.05). Bars display mean ± SE.

Scheme 1:

Separation of measured 8-iso-PGF_2α levels into the fractions formed by PGHS and CLP using the 8-iso-PGF_2α / PGF_2α ratio.