"Natural Chlorophyll but Not Chlorophyllin Prevents Heme-Induced Cytotoxic and Hyperproliferative Effects in Rat Colon"

[Amazoniac]

Natural Chlorophyll but Not Chlorophyllin Prevents Heme-Induced Cytotoxic and Hyperproliferative Effects in Rat Colon

"Chlorophyll (Fig. 1B) is the ubiquitous pigment in green leafy vegetables; like heme, it also has a planar porphyrin backbone. Chlorophyll is different from heme mainly by having the nonreactive magnesium instead of the highly reactive transition metal iron in the center of the porphyrin. In addition, chlorophyll has an esterified phytol tail instead of a propionic side chain. We speculated that chlorophyll “sandwiches” heme to form hydrophobic heme-chlorophyll complexes; as a result, it blocks radical-mediated heme metabolism and the heme-induced changes in epithelial cell turnover (15). In this study, we wanted to investigate whether this protective effect of natural chlorophyll is due to its porphyrin ring or requires the intact phytol-conjugated molecule. Therefore, we compared the possible protective effect of chlorophyllins with that of natural chlorophyll."

"Chlorophyllins (Fig. 1C) are molecular analogs of chlorophyll studied for cancer prevention in vitro and in vivo because they might mimic the effects of chlorophyll (16–19). Chlorophyllins are food-grade molecules derived from chlorophyll. They are hydrophilic, due to hydrolysis of the phytol tail, and the magnesium in the center of the porphyrin ring is removed or replaced by another metal. In contrast to the limited in vivo studies with natural chlorophyll (20,21), chlorophyllins have received much more attention. Several studies indicate that chlorophyllins may have anticarcinogenic effects because their porphyrin macrocycle can either scavenge free radicals or form a complex with planar aromatic carcinogens and thus reduce their bioactivity (17,22,23)."

"This study shows that chlorophyllins, in contrast to chlorophyll, do not inhibit the heme-induced carcinogenic effects in rats."

"The chlorophyllins supplemented to our purified heme diets showed only a negligible inhibition of the heme-induced luminal cytotoxicity. This slight inhibition did not prevent the heme-induced changes in colonic cell turnover. In contrast, chlorophyll added to this heme diet inhibited all the heme-induced changes in luminal cytotoxicity and cell turnover."

"Most of the heme ingested is delivered to the large bowel (31,32). There, a variable amount is converted to a range of iron-free porphyrins such as protoporphyrin, deuteroporphyrin, and pemptoporphyrin as a result of bacterial action (32). However, further degradation products such as di- and tripyrroles were not identified. The heme-induced cytotoxicity observed in our experiments results from the presence of a highly cytotoxic heme metabolite. This is a lipid-soluble, covalently modified porphyrin formed in the gut lumen of rats (7,30). Table 2 shows that chlorophyll, unlike the chlorophyllins, allowed most of the heme ingested to reach the feces without modification. This indicates that chlorophyll, but not chlorophyllin, prevents intestinal heme metabolism."

"Our previous work showed that the presence of the heme metabolite in the colonic lumen damages the colon surface epithelial cells and consequently increases epithelial proliferation and inhibits apoptosis in the colonic crypts (15). Spinach or an equimolar amount of chlorophyll prevented this heme-induced effect (15). We hypothesized that this is due to a “sandwich” of heme with chlorophyll molecules. As a result, chlorophyll may block the chemical reactivity of heme and thus the formation of its cytotoxic metabolite. This “sandwich” could be due to pi-pi interactions between heme and chlorophyll in a hydrophobic complex, analogous to the mechanism described by Dashwood et al. (33) for the interaction between chlorophyllins and planar aromatic compounds, such as heterocyclic amines."

"We showed previously that the detrimental effects of heme coincide with heme-catalyzed lipid peroxidation in the gut lumen (7) and that both are inhibited by dietary antioxidants (12). This implies that heme has to be in close contact with fatty acids in the hydrophobic phase of the luminal contents. We now show that, in contrast to chlorophyllins, the addition of chlorophyll to a heme diet decreased this heme-induced lipid peroxidation (Fig. 4). This indicates that the chlorophyllins we used in our model cannot “sandwich” heme to inhibit a reaction between heme and fatty acids in the diet. Only chlorophyll might be able to “sandwich” heme and as a consequence inhibit the catalytic activity of heme in the generation of lipid hydroperoxides and the formation of a cytotoxic heme metabolite (39). The difference in hydrophobic behavior of chlorophyll and chlorophyllin is a consequence of a structural difference. Chlorophyll is extremely hydrophobic due to the presence of a phytol tail, which is retained during intestinal passage (35,40). On the other hand, chlorophyllins are hydrophilic as a result of the removal of the phytol tail from chlorophyll."

"[..]heme and chlorophyllins are negatively charged under physiologic conditions such as prevail in the intestines, whereas chlorophyll remains a neutral molecule. These negative charges cause repulsive forces between heme and chlorophyllin, which may prevent formation of a complex between these molecules."