Document: HEI-3-42-17

True-blue, or blue and untrue? Use and misuse of Folin assays of polyphenols in ecological studies.

APPEL, H.M.* ¹, H.L.GOVENOR ², M.D'ASCENZO ³, E.SISKA ⁴ and J.C.SCHULTZ ¹

Penn State University, University Park PA 16802 ¹
Michigan State University, East Lansing MI 48824 ²
Boyce Thompson Institute for Plant Science, Ithaca NY, 14853 ³
Brown University, Providence RI 02912 ⁴

Abstract:
Ecologists frequently use colorimetric redox reagents (Folin-Denis, Folin-Ciocalteau, Prussian Blue) to estimate amounts of "total phenolics". However, the color of these reagents is influenced not only by the amount of phenolics present, but also by their structural variation and by non-phenolic reducing agents. Even if two samples have the same amount of phenolics, their Folin-Denis values may differ due to structural variation. We evaluated the importance of structural variation to the Folin-Denis concentration estimates of purified tannins from 16 woody plant species and 3 commercial polyphenol preparations. For each species, we generated regressions of Folin-Denis absorbances for a dilution series of purified tannin. The slopes of these relationships differed significantly among tree species, commercial preparation, and tree sampling dates. Because of this variation, each Folin absorbance indicates concentration only for a given species or sample and absorbances cannot be compared among them. Using a common standard worsens the problem. Using commercial 'tannins' as standards produced estimates ranging from 40% to 210% of the known concentrations, depending on the tree species. Thus, redox reagents such as the Folin-Denis produce reliable estimates of phenolic concentrations only when standards of purified polyphenols are prepared from the samples of interest. This requires preparing separate standards from each species, each tissue (e.g., leaf age), on each sampling date. We suggest that many published ecological studies testing hypotheses about "investment in defense", "cost of defense", etc. are highly suspect if they depended on these redox assays. With appropriate standards, redox reagents can be useful in ecological studies as indicators of redox-based biological activity and as indicators of relative amounts of phenolics. However, redox reagents should not be used to quantify absolute amounts of foliar polyphenols, or to make interspecific or temporal comparisons using a common standard. We provide recommendations for maximizing the utility of these assays and avoiding misinformation.

Keywords: phenolics, folin denis, tree defenses, tannins

This abstract is being presented at: 8:15 AM in session:
Oral Session #44: Terrestrial Invertebrates: Foodwebs and Plant Responses.