Distribution and sources of cyclic pheophorbides in the marine environment

Abstract

The labile chlorophyll a (Chl a 1 ) degradation product, 13 2 , 17 3 -cyclopheophorbide a enol (CPP516), was only recently found in marine sediments, where it can contribute a significant fraction to solvent-extractable sedimentary chlorins. We developed a new chromatographic method for the analysis of CPP516 to study its distribution and sources in the marine environment. We found high concentrations of CPP516 in surficial sediments and particulate matter collected in sediment traps. It contributed 40 to 70% to the sum of all Chl a 1 degradation products in fecal material from herbivorous microzooplankton and macrozooplankton. We did not find CPP516 in exponentially growing or stationary phase algal cultures. These data suggest that a major source of CPP516 is herbivores feeding on phytoplankton. We also found a series of compounds similar to CPP516 in sediments and sediment-trap samples. Mass-spectrometric and ultraviolet(UV)/visible-spectrometric analyses suggest that these are CPP516 analogs of divinyl-Chl a and Chl bs and Chl cs, conclusions that have to be confirmed by synthesis. CPP516, dissolved in organic solvents, easily degrades to 13 2 -oxopyropheophorbide a and chlorophyllone a under oxic conditions. When associated with particles, CPP516 is stable in oxic aquatic environments on a timescale of days, possibly stabilized by the chelation of metal cations. The chromophore of CPP516 differs substantially from that of other Chl a 1 degradation products; it has absorption maxima at 360, 426, and 688 nm. CPP516 does not fluoresce as other Chl a 1 degradation products. As a consequence, it is invisible to fluorescence-based analyses of ''pheopigments.'' Thus, rates of grazing and growth derived from such measurements, as used in conjunction with the gut fluorescence and the Chl-budget method, will be underestimates of true rates when CPP516 contributes significantly to total chlorins.

On the sedimentary occurrence of chlorophyllone a

“…The data could be compatible with the 13 2 -oxopyropheophorbide a (5) but 5 should elute around 9 min in the HPLC conditions we used (c.f. Goericke et al, 2000). On the basis of retention time and UV/vis spectrum, we tentatively identify this compound as 15 1 -hydroxychlorophyllonelactone a (8) previously reported by Watanabe et al (1993) in marine bivalves, together with 2(R or S), and other oxidized chlorophyll a derivatives such as chlorophyllonic acid 7 and purpurin 18 (9) as well as its methyl ester (9 0 ).…”

“…For example, on a Merck RP-18 column (Lichrosphere, 250 Â 4 mm) using binary (acetone/methanol) or ternary (acetone/methanol/water) solvent systems as we use in our laboratory (Verzegnassi et al, 1999;Riffe e-Chalard et al, 2000) CPP was almost undistinguished from the baseline. With a shorter column (Alltech RP-18 Adsorbosphere, 150 Â 4.6 mm, 3 lm), and using a solvent system and gradient (see Section 2) similar to those reported by Goericke et al (2000), we have been able to obtain a satisfactory peak shape.…”

“…As pointed out by Goericke et al (2000), HPLC separation of CPP using ÔtraditionalÕ methods applied to tetrapyrrolic pigments is not possible. For example, on a Merck RP-18 column (Lichrosphere, 250 Â 4 mm) using binary (acetone/methanol) or ternary (acetone/methanol/water) solvent systems as we use in our laboratory (Verzegnassi et al, 1999;Riffe e-Chalard et al, 2000) CPP was almost undistinguished from the baseline.…”

“…Another compound with close structural resemblance to Chlone a, 13 2 ,17 3 -cyclopheophorbide a enol (CPP, 4) had also been isolated and characterized from a marine sponge (Karuso et al, 1986). CPP, probably a metabolic intermediate of Chlone a, was recently proved to be widely distributed in sediments by three different groups (Goericke et al, 2000;Louda et al, 2000;Ocampo et al, 2000). However, all three reports pointed out the unstability of CPP.…”

“…Degradation occurred even on RP-HPLC columns when mobile phase contained antioxidants or buffer solutions. Chemosphere 52 (2003) 937-942 www.elsevier.com/locate/chemosphere Goericke et al (2000) observed that CPP was also degraded in organic solvents, more particularly in the presence of air. Main degradation products were the two chlorophylone a isomers (chlone a and chlone a 0 in approximately equal amounts) and 13 2 -oxopyropheophorbide a (oxoPPde a, 5).…”