Pheophorbides Chlorophylls

Esterification increases the lipophilic character of the pigments that has been recogiuzed as an important factor for interactions with the peptide chains of proteins. The hydrolysis of this side chain results in chlorophyllides and the concomitant removal of the Mg + ion in pheophorbides. Only a Umited number of natural chlorophylls in plants and photosynthetic organisms has been described and is well... 

Hortensteiner, S. et ah. The key step in chlorophyll breakdown in higher plants Cleavage of pheophorbide a macrocycle by a monooxigenase, J. Biol. Chem., 273, 15335, 1998. 

Hortensteiner, S., Vicentini, F., and Matile, P., Chlorophyll breakdown in senescent cotyledons of rape, Brassica napus L. enzymatic cleavage of pheophorbide a in vitro, New Phytol, 129, 237, 1995. 

Not-senescent and fresh-cut plants are almost devoid of degradation products like pheophytins and pheophorbides because chlorophylls associated with caro-... 

FIGURE 4.1.1 Possible chlorophyll degradation pathways in plant tissues or in processed foods. Pheophorbide a monooxygenase is specific for pheophorbide a. RCC = red chlorophyll catabolite. FCC = fluorescent chlorophyll catabolite. NCC = non-fluorescent chlorophyll catabolite. 

In the past, no snitable analytical methodologies were capable of investigating these multiple reactions and even today, the complete extraction and analysis of all the componnds is still a difficult task. The methods for extraction must be optimized for each sample according to the solubility of either phytylated (chlorophylls and pheophytins) or dephytylated (chlorophyllides and pheophorbides) derivatives, often requiring several repeated steps and the use of a single or a mixture of organic solvents. 

Chlorophyll catabolism has been intensively studied in some plants, e.g., rape-seed, barley, spinach, tobacco, Cercidiphyllum japonicum, Lolium temulentum, Liq-quidambar styraciflua and Arabidopsis thaliana, which present all NCC catabolites with similar basic structures. " This suggests a uniform breakdown of chlorophyll in which the oxidative opening of pheophorbide a seems to be a key step. Structural differences among the compounds have been related to at least six basic types of peripheral transformations. Some of them seem to operate either in sequence or in parallel, depending on the plant species, which caused the appearances of different... 

However, this accumulation has not been unequivocally proven. The recent identihcation of urobilinogenoidic linear tetrapyrroles in extracts from primary leaves of barley indicated that further degradation of the v-NCC 1 can take place. While the monoxygenation of pheophorbide a in the earlier phases of chlorophyll breakdown in higher plants appears to be a remarkably stringent entry point, the rather diverse structures of NCCs may indicate that the later phases of the detoxi-hcation process follow less strictly regulated pathways." ... 

The development and reports of methods for colorless chlorophyll derivative (RCCs, FCCs, and NCCs) analysis are relatively recent and the structures of the compounds are being elucidated by deduction from their chromatographic behaviors, spectral characteristics (UV-Vis absorbance spectra), mass spectrometry, and nuclear magnetic resonance analysis. The main obstacle is that these compounds do not accumulate in appreciable quantities in situ and, moreover, there are no standards for them. The determination of the enzymatic activities of red chlorophyll catabolite reductase (RCCR) and pheophorbide a monoxygenase (PAO) also helps to monitor the appearance of colorless derivatives since they are the key enzymes responsible for the loss of green color. ... 

Usually, HPLC analysis resolves four peaks identified by co-chromatography with authentic standards as copper pheophorbide a, Cn(II) chlorin e6, Cn(II) chlorin e4, Cu rhodin g7, and their degradation products, but a sum of other colored components can also be found, for example, native chlorophylls, pheophytins, pheophor-bides, and rodochlorins (free carboxyl forms of pheophorbides) besides epimers, allomers, and degradation products that have been only tentatively identified. 

Chlorophyll-a Pheophytin-a Pyropheophitin- a Pheophorbide-a Pyropheophorobide-a Isofucoxanthin-dehydrate Fucoxanthin dehydrate Fucoxanthin-hemiketal Isofucoxanthin dehydrate pheophorbide a ester Isofucoxanthin dehydrate pheophorbide a ester Isofucoxanthin dehydrate pyropheophorbide a ester 23.5 26.4 28.1 5.0 6.9 10.7 12.0 6.4 24.4 22.9 25.4... 

Fig. 2.130. Elution profile by RP-HPLC of the chlorophyll derivative pigments analysed. The pigments were detected spectrophotometrically at 660 nm and fhiorimetrically using excitation and emission wavelengths at 440 and 660 nm, respectively. Peak identification (numbers in parentheses are retention times in min) 1 = chlorophyllide-b (3.10) 2 = chlorophyllide-a (4.98) 3 = pheophorbide-b (7.44) 4 = pheophorbide-a (8.85) 5 = chlorophyll-b (14.74) 6 = chlorophyll-a (16.40) 7 = pheophytin-b (21.49) 8 = pheophytin-a (23.38). Reprinted with permission from L. Almela et al. [301].

The vinyl groups can be retained in many of the degradations of chlorophylls thus 3-vinylpheoporphyrin a5 (106) and 3-vinylrhodoporphyrin-XV (107) can be obtained. Both chlorin e6 and rhodin gy can be re-converted by treatment with methoxide or, better, with butoxide (8OJOC2218) into pheophorbides a or b, respectively. Of the three carboxylic acid groups in chlorin e6, those at the methine and 13-positions can be removed thermally to give chlorin e4 (108) and isochlorin e4 (109), together with phyllochlorin (110). 

Resuspension of bottom sediments presents a potential problem for flux estimates. However, our results suggest minimal resuspension during stratification. As a part of a separate study, Hurley (unpublished data) measured pigment fluxes to the sediment surface. Sediment trap material was dominated by chlorophyll a and pheophorbide a (a grazing indicator). Surface sediments, however, were dominated by pheophytin a, a relatively stable chlorophyll degradation product. The lack of any substantial amounts of pheophytin in trap material suggested that if resuspension of particulates from the surface sediment was important, it was probably minimal. 

Phenylspiro[furan-2(3 H), 1 -phthalan]-3,3-dione. see Fluorescamine Pheophorbides. see also Chlorophylls Pheophytins. see also Chlorophylls Phospholipids, 523... 

Development of fast, accurate, and reproducible high-performance liquid chromatography (HPLC) methods has offset the use of traditional open-column and TLC methods in modern chlorophyll separation and analysis. A number of normal and reversed-phase methods have been developed for analysis of chlorophyll derivatives in food samples (unit F4.4), with octadecyl-bonded stationary phase (C]8) techniques predominating in the literature (Schwartz and Lorenzo, 1990). Inclusion of buffer salts such as ammonium acetate in the mobile phase is often useful, as this provides a proton equilibrium suitable for ionizable chlorophyllides and pheophorbides (Almela et al., 2000). 

White, R.C., Jones, I.D., and Gibbs, E. 1963. Determination of chlorophylls, chlorophyllides, pheo-phytins and pheophorbides in plant material. J. FoodSci. 28 431-436. 

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