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Background: Excess 5-aminolevulinic acid (ALA) and α-aminoacetone (AA) are implicated in ketosis, porphyrinpathies and diabetes. Pathologic manifestations involve O₂⁻, H₂O₂, OH, enoyl radicals (ALA and AA) and their oxidation end products., Methods: To characterize enoyl radicals resulting from reaction of OH radicals with ALA and AA, micromolar OH concentrations were produced by pulse radiolysis of ALA and AA in aqueous solutions., Results: ALA and AA react with OH at k=1.5 × 10⁹ M⁻¹s⁻¹. At pH7.4, the ALA absorbance spectrum has a maximum at 330 nm (ε=750 M⁻¹cm⁻¹). This band appears as a shoulder at pH8.3 where two ALA species are present: (NH3)⁺-CH₂-CO-CH₂-CH₂-COO⁻ and NH₂-CH₂-CO-CH₂-CH₂-COO⁻ (pKa=8.3). At pH8.3, ALA reacts with oxygen (k=1.4 × 10⁸ M⁻¹s⁻¹) but not with O₂⁻. At pH8.3, AA oxidation produces two AA species characterized by an absorbance spectrum with maxima at 330 and 450 nm. ALA and AA are repaired by antioxidants (quercetin (QH), catechin, trolox, ascorbate) which are semi-oxidized (k>10(8)M⁻¹s⁻¹). QH bound to HSA or to apoferritin and ferritin repairs ALA and AA. In O₂-saturated apoferritin solutions, Q, O₂⁻, AA and reaction product(s) react with QH., Conclusions: The optical absorption properties and the time evolution of ALA and AA were established for the first time. These radicals and their reaction products may be neutralized by antioxidants free in solution or bound to proteins., General Significance: Adjuvant antioxidant administration may be of interest in pathologies related to excess ALA or AA production., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Neutral tryptophan (*Trp) and tyrosine (TyrO(*)) radicals are repaired by certain flavonoids in buffer, in micelles and in human serum albumin (HSA) with corresponding formation of semioxidized flavonoid radicals. In deaerated buffer, *Trp but not TyrO(*) radicals react with catechin. In micelles, quercetin and rutin repair both *Trp and TyrO(*) radicals. In addition to amino acid reactivity, microenvironmental factors and nature of the flavonoids govern this repair. Electron transfer efficiencies from quercetin to negatively charged *Trp radicals are 100% in the micellar pseudophases of positively charged cetyltrimethylammonium bromide, (CTAB), and neutral Triton X100 (TX100), but 55% in negatively charged sodium dodecyl sulfate (SDS). In oxygen-saturated CTAB micelles, quercetin also reacts with the superoxide radical anion. When bound to domain IIA of HSA, quercetin repairs, by intra- or intermolecular encounter, less than 20% of oxidative damage to HSA. Quercetin can also repair freely circulating oxidized molecules with repair efficiencies falling to 7% for oxidized Trp, Tyr and alpha-MSH and to less than 2% for urate radical. This limited effectiveness is attributed both to the inaccessibility of bound quercetin and rutin toward radicals of circulating molecules and to the diffusion-controlled recombination of these radicals.

Neutral tryptophan (*Trp) and tyrosine (TyrO(*)) radicals are repaired by certain flavonoids in buffer, in micelles and in human serum albumin (HSA) with corresponding formation of semioxidized flavonoid radicals. In deaerated buffer, *Trp but not TyrO(*) radicals react with catechin. In micelles, quercetin and rutin repair both *Trp and TyrO(*) radicals. In addition to amino acid reactivity, microenvironmental factors and nature of the flavonoids govern this repair. Electron transfer efficiencies from quercetin to negatively charged *Trp radicals are 100% in the micellar pseudophases of positively charged cetyltrimethylammonium bromide, (CTAB), and neutral Triton X100 (TX100), but 55% in negatively charged sodium dodecyl sulfate (SDS). In oxygen-saturated CTAB micelles, quercetin also reacts with the superoxide radical anion. When bound to domain IIA of HSA, quercetin repairs, by intra- or intermolecular encounter, less than 20% of oxidative damage to HSA. Quercetin can also repair freely circulating oxidized molecules with repair efficiencies falling to 7% for oxidized *Trp, Tyr and alpha-MSH and to less than 2% for urate radical. This limited effectiveness is attributed both to the inaccessibility of bound quercetin and rutin toward radicals of circulating molecules and to the diffusion-controlled recombination of these radicals.

The influence of the calcium channel blocker verapamil on the sensitivity of mouse fibrosarcoma cells of the line EMT-6 to treatment with Photofrin II (PII) or tetra(4-sulfonatophenyl)porphine (TPPS4) and light has been assessed. Cells were treated with 1.5 microg/ml PII or 75 microg/ml TPPS4 overnight in the absence or presence of 50 microg/ml verapamil and subsequently exposed to light. Verapamil increased the sensitivity of the EMT-6 cells to PII-induced photoinactivation by a factor of 2. In contrast, verapamil decreased the sensitivity of the cells to TPPS4-induced photoinactivation by 50-60%. Both sensitizers were found to be located to a large extent in lysosomes as revealed by fluorescence microscopy and by photochemical inactivation of the lysosomal marker enzyme beta-N-acetyl-D-glucosaminidase. Verapamil increased the uptake of PII by 30% and reduced the uptake of TPPS4 by 20%. Furthermore, verapamil enhanced the binding and uptake of LDL by about 40%. In conclusion, the effects of verapamil-induced sensitization of EMT-6 cells treated with PII or TPPS4 and light can to a large extent be attributed to the modulatory effects of verapamil on endocytosis., (Copyright 1998 Elsevier Science B.V.)

Human skin chronically exposed to UV light is known to accumulate iron and to have an increased ferritin content as compared to unexposed areas. Iron accumulation is also found in many inflammatory skin diseases. Cultured human fibroblasts loaded with iron by incubation with non-toxic concentrations of the ferric nitrilotriacetate complex have been irradiated with low (up to 15 J/cm2) and moderate (up to 45 J/cm2) UVA doses. At low irradiation doses, lipid peroxidation doubles without affecting the viability of iron-loaded cells. At higher irradiation doses (30 J/cm2) the photocytotoxicity of UVA towards iron-loaded cells increases in a concentration-dependent manner with the iron load. Thus, after exposure to 30 J/cm2 of UVA, the cytotoxicity is about 3-fold greater for cells incubated for 75 min with 100 microM of the ferric complex as compared to those not treated with the ferric complex. Incubation with desferrioxamine, an extremely efficient chelator of ferric ion or vitamin E, a radical scavenger which blocks the lipid peroxidation radical chain, leads to marked inhibition of the sensitizing effects of iron on lipid peroxidation but is less effective for the survival of cells exposed to UVA. A similar concentration-dependent protective effect of desferrioxamine was observed with cultured fibroblasts not treated with the ferric complex. It is suggested that the photoreduction of ferritin and/or other iron-containing proteins plays a significant role in the UVA-induced photocytotoxicity of skin fibroblasts.

The ultraviolet B-induced destruction of tryptophan residues and lipid peroxidation of high-density lipoproteins is accompanied by the immediate and marked structural modification of the apolipoproteins, as revealed by SDS-polyacrylamide gel electrophoresis and immunoblot with specific monoclonal antibodies. Formation of several polymers of apolipoprotein A-I, apolipoprotein A-II or both apolipoproteins occurred, although apolipoprotein A-II did not contain any Trp residue. These results suggest that initial photochemical damage can be transferred via intramacromolecular processes to other sites within the same apolipoprotein and by intermacromolecular reactions from apolipoprotein A-I to other apolipoproteins. In both cases, lipid peroxidation enhances the propagation of the initial photochemical damage. The physiological significance of this work is discussed with respect to the low-light doses required for the alterations of the high-density lipoproteins.

Malondialdehyde modification and copper ion-induced autooxidation of the apo E-free HDL3 fraction of high-density lipoproteins were studied with respect to physico-chemical characteristics and physiological properties of the lipoprotein. Cu(2+)-oxidized HDL was much less modified than MDA-treated HDL, in terms of electrophoretic mobility, lipid peroxidation product content, Lys and Trp amino acid residue level and polymerization of apo A-I. With [3H]cholesteryl linoleate-labeled LDL, an inhibition of cholesterol efflux was observed in the presence of modified HDL, with a more marked effect with MDA-modified HDL. Competition studies with iodinated native HDL demonstrated a decreased binding of modified HDL to cell surface receptors. The decrease in cholesterol intracellular content, determined either by the isotopic equilibrium method or by the enzymatic cholesterol oxidase technic, was less marked in the presence of modified HDL than in the presence of native HDL. MDA-modified HDL was the less effective in decreasing cellular cholesterol content. It is thus suggested that malondialdehyde-induced alteration of HDL, or HDL peroxidation, if occurring in vivo, could contribute to the progress of atherogenesis by decreasing cholesterol efflux from peripheral tissues.

The tryptophan residues of HDL are important chromophores of the interstitial fluid feeding the epidermal cells. The UVB light readily photoionizes tryptophan residues as demonstrated by the increase in their photolysis yield in presence of N2O. Saturation of the HDL solution with a N2O/O2 mixture (80%:20%, v/v) decreases the peroxidation of HDL lipids, thereby implying that lipid peroxidation is at least partly induced by tryptophan photolysis. Addition of EDTA and desferrioxamine to HDL solutions loaded with either Cu2+ or Fe2+ ions suggests that Fe2+ could be the 'contaminating' trace metal ions that are required to explain the occurrence of lipid photoperoxidation induced by photons absorbed by tryptophan residues.

The UVA irradiation of cultured human fibroblasts leads to the formation and to the release of thiobarbituric acid-reactive substances in the supernatant. The major thiobarbituric acid-reactive substance is identified by fluorescence spectroscopy and HPLC, as malondialdehyde or malondialdehyde-forming substances under the thiobarbituric acid assay conditions. Malondialdehyde formation strongly suggests a UVA-induced lipid peroxidation. Lipid peroxidation is also supported by the inhibitory effect of D,L-alpha-tocopherol, the well-known chain breaking antioxidant, by the additional malondialdehyde formation in the dark after the photooxidative stress and by membrane damage revealed by lactate dehydrogenase leakage.

Serum lipoproteins are good carriers for the aromatic retinoid Ro 10-9359 (etretinate) and to a lesser extent for its main metabolite in human Ro 10-1670 (acitretin). Up to about 200 and 130 etretinate molecules and 200 and 70 acitretin molecules can bind to one LDL and one HDL, respectively. In contrast human serum albumin only binds about 10 etretinate or 30 acitretin molecules. In whole human serum loaded with the retinoids, lipoproteins carry approx. 67% of total etretinate or approx. 37% of total acitretin. In the particular case of etretinate, low density lipoproteins account for about 30% of the lipoprotein-carried etretinate.

Etretinate or acitretin are efficiently delivered to cultured human fibroblasts in the presence of low density lipoproteins, high density lipoproteins or human serum albumin. In contrast to acitretin, delivery of etretinate to fibroblasts is more efficiently achieved with human serum albumin than with lipoproteins. The uptake of etretinate and acitretin via low density lipoproteins delivery, does not take place via the low density lipoprotein-receptor endocytotic pathway but mostly through a passive exchange with the plasma membrane. However, in contrast to acitretin, the exchange of etretinate seems to occur alter binding of etretinate-loaded low density lipoproteins to the apolipoprotein B receptors. No differences are observed in binding, internalization and degradation of native, etretinate-loaded low density lipoproteins and acitretin-loaded low density lipoproteins, suggesting that the presence of these retinoids in low density lipoproteins does not alter their processing by the cells. Furthermore, the presence of these retinoids in the cells does not notably affect, under our experimental conditions, the catabolism of native low density lipoproteins.

Tryptamine, serotonin and tryptophan are readily oxidized during the Cu2+-catalyzed peroxidation of arachidonic acid (AA) at neutral pH and under certain experimental conditions which determine their relative susceptibility to oxidation. Thus, in AA micelles, fluorescence spectroscopy demonstrates that positively-charged indoles interact with negatively-charged micelles while Trp remains in the aqueous phase. As a result, serotonin and tryptamine are preferentially oxidized. In egg phosphatidylcholine liposomes loaded with AA, the three substrates interact with vesicles and undergo lipid-induced oxidation. EDTA inhibits the formation of thiobarbituric-reactive substances (TBARS) and prevents the indoles from oxidation. Owing to the intricate contact between the lipidic core and the apolipoproteins, the Trp residues of human serum LDL and HDL3 are very rapidly oxidized, i.e., at least one order of magnitude faster than Tyr HDL and Lys LDL, which are believed to be involved in the binding of these lipoproteins to their cell receptors. Cupric ions are rather specific for the lipid-induced autoxidation of Trp residues of lipoproteins whereas in micelles and liposomes, Mn2+ and Fe2+ can lead to TBARS production and to oxidation of indoles. This specificity is surprising considering the known ability of Fe2+ to catalyze LDL modification (measured by TBARS production) during their incubation with various cells. Biological consequences of the easy lipid-induced oxidation of biologically important indoles are discussed.

Microspectrofluorometry of L and WI-38 cells reveals chemical/structural changes due to quiescence or senescence, i.e., lipid peroxidation, spontaneous or photosensitized by hematoporphyrin. Cells treated with hematoporphyrin and a lysosomal umbelliferone probe show a fast-rising umbelliferone emission, plus a fluorescent photoproduct. Studies in rapidly growing versus quiescent L, early passage/late passage WI-38 cells, suggest accumulation of fluorescence Schiff bases (i.e., their association with granular regions of cells in stationary phase, spectral properties, fast increase in photosensitized cells) and a possible lysosomal membrane permeabilization in quiescent or senescent cells.

Resonance energy transfer from tyrosine to tryptophan residues was detected in the phage fd. The magnitude of the transfer efficiency was estimated by both a traditional and an alternative method. The latter involved comparison of the indole acceptor excitation spectrum full-width half-maximum with a set of standard values differing in the amount of absorbance contributed by tyrosine donor. Both methods lead to the same conclusion: essentially all the tyrosine residues of the viral coat are within 0.9 nm of a tryptophan residue. Also, fluorescence lifetime measurements provide additional support for the hypothesis that there are at least two different environments for the coat protein's sole indole side-chain. Little if any DNA phosphorescence was seen, consistent with the nucleic acid bases being stacked in the DNA core.

Charge transfer has been observed between oxidised tryptophan-26 units and the tyrosine-21 or -24 of the coat protein of fd phage. The transfer is likely to be intramolecular. The rates suggest that the aromatic units are in a rigid region and that they may have at least two different environments. No apparent interaction occurs with the DNA, consistent with tryptophan and tyrosine units not being in contact with the bases.

The decay of the indole triplet of single tryptophan-containing proteins and model compounds can be readily determined at room temperature in solution by monitoring the triplet absorption or emission following an exciting laser pulse. The dioxygen triplet quenching constants, can be measured for all these molecules and compared to the analogous singlet values determined by fluorescence methods. The dioxygen triplet quenching constant (tkq) ranged from a high of 5.1.10(9) M-1.s-1 for the exposed indole of corticotropin to a low of 0.1.10(9) M-1.s-1 for the buried indole of asparaginase. The ratio of these values with their respective dioxygen singlet quenching constants (skq), tkq/skq, ranged from 0.3 to 0.6 for aqueous exposed polypeptide indoles. For globular proteins the tkq/skq value is observed to be 0.2 +/- 0.1. This lower value for protein indoles is not attributable to 'bulk' environmental or hydrogen bonding effects, since the magnitude of tkq/skq (= 0.5 +/- 0.1) for model indoles was independent of solvent dielectric constant, polarity, and proticity. Temperature-dependence studies were done to test whether tkq could be used to characterize the nature of the protein matrix. The activation energy (Ea) for tkq was found to be 11 +/- 2 kcal/mol for most proteins. This Ea was independent of whether the indole side-chain was solvent exposed or buried in the non-aqueous protein interior. Large Ea values were also obtained for model indoles, naphthalene and nalidixic acid, dissolved in water, whereas the same compounds dissolved in 95% ethanol exhibited much smaller Ea values. These data, in combination with the observation that the tkq of model indoles is insensitive to changes in solvent viscosity, indicate that dioxygen quenching at the triplet level can not be easily used to characterize the dynamics of proteins.