Kinetics and thermodynamics of lipid amphiphile exchange between lipoproteins and albumin in serum.

We have examined the kinetics and thermodynamics of the exchange of a fluorescent amphiphile derived from a phospholipid, NBD-DMPE, between serum albumin and the serum lipoproteins of high density (HDL2 and HDL3), LDL, and VLDL. Binding of the fluorescent lipid amphiphile to bovine serum albumin is characterized, at 35 degrees C, by an equilibrium binding constant of approximately 3 x 10(6) M(-1) and a characteristic time < or = 0.1 s. Association of NBD-DMPE with the lipoprotein particles, if considered as a partitioning of amphiphile monomers between the aqueous phase and the lipoprotein particles, is characterized by an equilibrium partition coefficient between 10(5) and 10(6), being highest for LDL and lowest for HDL. The association of NBD-DMPE monomers with lipoprotein particles can be described by insertion rate constants on the order of 10(5) M(-1) s(-1) for VLDL and LDL and 10(4) M(-1) s(-1) for HDL. The desorption rate constants are on the order of 10(-5) s(-1) for all particles. The study was performed as a function of temperature between 15 and 35 degrees C. This permitted the calculation of the equilibrium thermodynamic parameters (deltaG(o), deltaH(o), and deltaS(o)) as well as the activation parameters (deltaG++(o), deltaH++(o), and deltaS++(o)) for the insertion and desorption processes. The association equilibrium is dominated by the entropic contribution to the free energy in all cases. The results are discussed in relation to phospholipid and amphiphile exchange phenomena involving the lipoproteins.

Sulfonated chloroaluminum phthalocyanine incorporates into human plasma lipoproteins: photooxidation of low-density lipoproteins.

The interactions of sulfonated chloroaluminum phthalocyanine (AlPcSn) with human low-density lipoproteins (LDL) were studied in vitro in human plasma and in an isolated LDL fraction, in order to understand the potential effects of the sensitizer against LDL. The AlPcSn added to plasma distributes in all lipoproteins as observed by the drastic color changes of the separated fractions by ultracentrifugation. In isolated LDL, incubation with AlPcSn causes fluorescence quenching of the apoprotein tryptophan residues. Furthermore, AlPcSn incorporates in liposomes, with a lipid composition similar to the external monolayer of human LDL, as indicated by absorbance spectroscopy. The photosensitizing properties of AlPcSn in LDL particles were studied on the basis of the fluorescence quenching of previously incorporated cis-parinaric acid (PnA), used as an oxidation probe, and of O2 consumption. The photooxidation of either PnA or LDL lipids is highly dependent on irradiation time and on the dye concentration. Moreover, photooxidation of LDL proceeds only during the illumination period. After stopping the illumination and upon addition of Cu2+ to the LDL solution, the oxidative rate is resumed, probably due to hydroperoxide cleavage and formation of species able to propagate the oxidative reaction. Thus, our data indicate that AlPcSn distributes in human plasma lipoproteins and, in isolated LDL, it can interact either with the lipid phase or the apoprotein. The photooxidation of LDL induced by AlPcSn seems to involve singlet oxygen as the main reactive species in the degradative process.

Rapid isolation of low density lipoproteins in a concentrated fraction free from water-soluble plasma antioxidants.

A rapid method is described for isolation and concentration of plasma low density lipoproteins (LDL) using a Beckman L80 ultracentrifuge equipped with a 70.1 Ti fixed angle rotor. The isolation of LDL achieved by a discontinuous gradient density step (180 min) was followed by a simultaneous purification and concentration step (45 min) using ultrafiltration through a collodium bag under nitrogen. This dialysis/concentration step, in contrast to the standard dialysis techniques in batch or by filtration through short gel columns, prevents oxidation and dilution of the sample. Electrophoresis in agarose and sodium dodecylsulfate-polyacrylamide (SDS-PAGE) gels were used to monitor LDL surface charge, purity, and contamination with plasma proteins. The artifactual oxidation of LDL during isolation and subsequent handling, and thus the ability of LDL preparation for oxidation/antioxidation studies, was assessed by the determination of endogenous hydroperoxides and thiobarbituric acid reactive substances. The dialysis/concentration step by ultrafiltration that allows the obtention of a concentrated and purified LDL preparation was validated by the absence of ascorbate and urate, as measured by HPLC. This method led to LDL preparations free of water-soluble plasma antioxidants that were minimally oxidized and suitable for reliable in vitro LDL oxidation and inhibition studies. The applicability of this methodology was tested by studying the alpha-tocopherol content of LDL in a Portuguese population of university students.

Reactivity of dietary phenolic acids with peroxyl radicals: antioxidant activity upon low density lipoprotein peroxidation.

The interaction of four phenolic acids, representative of three chemical groups present in human diet, with peroxyl radicals was studied in vitro in a low density lipoprotein (LDL) oxidation model. The controlled oxidation of LDL was initiated by free radicals generated from a hydrophilic azo initiator and followed by monitoring the oxygen consumption and the fluorescence quenching of cis-parinaric acid previously incorporated into LDL. The hydroxycinnamic acid derivatives, chlorogenic and caffeic acids, have high stoichiometric numbers and reactivity with peroxyl radicals as compared with trolox, the water-soluble analogue of vitamin E, whereas ellagic acid (a tannic compound) compares with trolox effects. Protocatechuic acid (a hydroxybenzoic acid derivative) exhibits a complex reaction with peroxyl radicals, as indicated by UV spectroscopy, resulting in undefined inhibition periods of LDL oxidation and low reactivity with peroxyl radicals. Presumably, secondary radicals of these compounds are unable to initiate LDL oxidation. The antioxidant activity of the various phenolic compounds is discussed in terms of structure-activity relationships.

Lipid peroxidation and its inhibition in low density lipoproteins: quenching of cis-parinaric acid fluorescence.

The fluorescent polyunsaturated parinaric acid incorporated in LDL particles is highly sensitive to the concentration of peroxyl radicals in the aqueous medium, undergoing rapidly oxidative degradation, as detected by a quenching of fluorescence, without delay after radical generation in solution. Ascorbate, cysteine, and urate suppress the parinaric acid fluorescence decay promoted by peroxyl radicals generated at a constant rate (thermal decomposition of 2,2'-azo-bis(2-amidino-propane hydrochloride)) in a concentration-dependent manner. The chain-breaking efficiencies of these antioxidants are evaluated from the time interval (inhibition period) of parinaric acid protection from oxidative degradation. The results correlate with the inhibition periods of LDL oxidation as monitored by O2 consumption. Therefore, the sensitive and simple parinaric acid assay can be used as a semiquantitative screening test for the detection of potentially important water-soluble chain-breaking antioxidants. Conversely to O2 consumption, the absence of any initial lag phase of probe degradation attests to the sensitivity of the assay. An improved methodology based on second-derivative spectroscopy to follow the formation of conjugated diene isomers directly in the preparation without the need for lipid extraction also confirms the sensitivity of this assay. To assess the usefulness of parinaric acid assay, strong chain-breaking activities of caffeic and chlorogenic acids are reported.