Article Figures & Data
Figures
- Fig. 1.
Free radical-catalyzed formation of phytoprostanes from linolenic acid. Hydrogen abstraction from one of the two bisallylic methylene groups of linolenic acid yields pentadienyl radicals that are prone to oxygenation and cyclization. Because the methyl and the carboxy chain (abbreviated with R) at the triene unit of linolenic acid are mechanistically equivalent, two regioisomeric PPG1 can be generated. The two regioisomers of the unstable PPG1 (each theoretically comprised of 16 stereomers) are precursors of type I and type II PPF1 (formed by reduction of both peroxy groups), as well as of PPE1 and PPD1(formed by rearrangement of the endoperoxy group followed by reduction of the hydroperoxy group).
- Fig. 2.
Formation of cyclic and acyclic trihydroxylated linolenic acid derivatives. Each cyclic triol structure (PPF1) shown is composed of 16 isomers. Acyclic triol structures formed by either autoxidation or photooxidation represent eight isomers. Under the conditions of photooxidation in vivo and in most in vitro systems, autoxidation cannot be prevented. Thus autoxidation products such as phytoprostanes cannot be used to differentiate between autoxidation and photooxidation. Acyclic triols produced via the peroxygenase or epoxy alcohol synthase pathway are single, enantiomerically pure isomers. The residuesR 1 and R 2represent CH3 and (CH2)6COOH, respectively.
- Fig. 3.
HPLC analysis of PPF1. PPF1 were identified in peaks labeled 1 to 7 and the regioisomeric composition (type I, type II, or both, as indicated in brackets) was established (see “Materials and Methods” section for details). Peaks labeled with (−) did not contain PPF1.
- Fig. 4.
MS of PPF1-type I and type II derivatives. PPF1 were analyzed as their corresponding methyl ester, TMS ether derivatives in the EI (spectra A and B) as well as in the CI (spectra C and D) mode. PPF1 were also analyzed as their PFB ester, TMS ether derivatives in the negative chemical ionization mode (spectra E and F). Type I compounds (A, C, and E) can only be differentiated from type II compounds (B, D, and F) by EI-MS. Characteristic cleavage points were indicated at the molecular formulae, see “Results” for details.
- Fig. 5.
GC-NICI-MS analysis of PPF1in dried peppermint leaves. PPF1 were determined as PFB ester, TMS ether derivatives without (A) and with (B) prior catalytic hydrogenation. Ion chromatograms at m/z 543 and 545 display endogenous PPF1 (20.01 ± 0.76 μg/g of dry weight), whereas m/z 549 and 551 display the triply oxygen-18 labeled internal standard. For quantification, peaks in the chromatograms of sample B were integrated together and the endogenous level of PPF1 was calculated from the ratio of the peak areas at m/z 545 to that at m/z551.
- Fig. 6.
Accuracy and precision of the quantitative analysis of PPF1. PPF1(1–1,500 ng) and [18O]3PPF1(500 ng, internal standard) were added to 170 mg (dry weight) of suspension culture cells of tobacco. PPF1 were extracted and analyzed as described in “Material and Methods.” Error bars indicate means ± sd (n = 3).
- Fig. 7.
Induction of PPF1 by oxidative stress and wounding. California poppy suspension cells were treated with 10 mm t-butyl hydroperoxide at zero time and levels of free (A) and esterified (B) PPF1 (●) were monitored. The time course of free (C) and esterified (D) PPF1 (●) in California poppy suspension cells was also monitored after application of 500 μm cupric acetate. The kinetic of free (E) and esterified (F) PPF1s was measured in wounded peppermint leaves (●). Peppermint leaves were cut off at the stem and wounded with a nail stamp or left intact. Leaves were incubated in a chamber with a water-saturated atmosphere. Levels of PPF1 (○) in untreated cells or leaves were determined in separate control experiments.
Tables
Sample PPF1 PPE1 Ratio of PPF1 to PPE1 μg/g Fresh leaves Free 0.076 ± 0.004 0.037 ± 0.005 2∶1 Esterified 11.24 ± 1.52 0.13 ± 0.004 86∶1 Autoxidized linolenate Protic solvent 2,300 5,600 0.41∶1 Aprotic solvent 11,700 300 39∶1 Levels of free and esterified phytoprostanes in peppermint leaves (micrograms per gram of dry wt.), as well as levels of phytoprostanes in autoxidized linolenate samples (micrograms per gram of linolenic acid) were determined by GC-MS. Analysis were performed in triplicate. Values are means ± sd (n = 3).
