Uri Cogan

Lovastatin inhibits low-density lipoprotein oxidation and alters its fluidity and uptake by macrophages: In vitro and in vivo studies

Under experimental conditions, oxidized low-density lipoprotein (Ox-LDL) may possess atherogenic properties, as is evidenced by its contribution to cholesterol accumulation in macrophages. LDL was oxidized in a cell-free system by predialysis of the lipoprotein against EDTA-free buffer and the addition of copper ions. Oxidation of LDL in the presence of lovastatin (10 to 1,000 mumol/L) resulted in a time- and dose-dependent reduction in thiobarbituric-acid-reactive substances (TBARS) concentration that was accompanied by increased LDL lysine-amino-group reactivity, in comparison with Ox-LDL produced in the absence of lovastatin. At 100 mumol/L, the drug reduced malondialdehyde concentration and increased amino-group reactivity by 24% and 42%, respectively. However, lovastatins antioxidant effect was limited relative to other antioxidants, such as probucol and vitamin E. The fluidity of Ox-LDL was substantially reduced in comparison with native LDL. However, lovastatin inhibited this reduction in fluidity by 20%. Upon incubation of J-774 macrophage-like cell line with Ox-LDL, the lovastatin-treated Ox-LDL induced a reduction in the cellular cholesterol esterification rate in comparison with the effect of Ox-LDL that was produced in the absence of the drug. In four patients with hypercholesterolemia, the effect of lovastatin therapy (20 mg/d) on the sensitivity of their LDL to in vitro oxidation was studied. In all patients, Ox-LDL prepared from LDL obtained during lovastatin treatment demonstrated a reduced TBARS content, an increased trinitrobenzenesulfonic acid (TNBS) reactivity, an increased fluidity, and an impaired uptake by macrophages. These results were similar to those obtained by adding lovastatin in vitro.

Aqueous Extracts of Teucrium polium Possess Remarkable Antioxidant Activity In Vitro

Teucrium polium L. (Lamiaceae) (RDC 1117) is a medicinal plant whose species have been used for over 2000 years in traditional medicine due to its diuretic, diaphoretic, tonic, antipyretic, antispasmodic and cholagogic properties. The therapeutic benefit of medicinal plants is often attributed to their antioxidant properties. We previously reported that an aqueous extract of the leaves and stems of this plant could inhibit iron-induced lipid peroxidation in rat liver homogenate at concentrations that were not toxic to cultured hepatic cells. Others have reported that organic extracts of the aerial components of this plant could inhibit oxidative processes. Against this background, we felt further investigation on the antioxidant action of the extract of T. polium prepared according to traditional Arab medicine was warranted. Accordingly, we assessed (i) its ability to inhibit (a) oxidation of β-carotene, (b) 2,2′-azobis(2-amidinopropan) dihydrochloride (AAPH)-induced plasma oxidation and (c) iron-induced lipid peroxidation in rat liver homogenates; (ii) to scavenge the superoxide (O2•−) radical and the hydroxyl radical (OH•); (iii) its effects on the enzyme xanthine oxidase activity; (iv) its capacity to bind iron; and (v) its effect on cell glutathione (GSH) homeostasis in cultured Hep G2 cells. We found that the extract (i) inhibited (a) oxidation of β-carotene, (b) AAPH-induced plasma oxidation (c) Fe2+-induced lipid peroxidation in rat liver homogenates (IC50 = 7 ± 2 μg ml−1); (ii) scavenged O2•−(IC50 = 12 ± 3 μg ml−1) and OH• (IC50 = 66 ± 20 μg ml−1); (iii) binds iron (IC50 = 79 ± 17 μg ml−1); and (iv) tended to increase intracellular GSH levels resulting in a decrease in the GSSG/GSH ratio. These results demonstrate that the extract prepared from the T. polium possesses antioxidant activity in vitro. Further investigations are needed to verify whether this antioxidant effect occurs in vivo.

The interaction between retinol-binding proteins and prealbumins studied by fluorescence polarization

The interaction between retinol-binding proteins and prealbumins of human and chicken was studied by fluorescence polarization techniques. The binding affinity between chicken plasma retinol-binding protein and chicken prealbumin was essentially the same as between the respective human proteins. Human urine retinol-binding protein displayed a similar affinity, though possibly slightly smaller than that of the human plasma protein, toward human prealbumin. Retinol-binding proteins and prealbumins of human and chicken have been found to cross-interact displaying an affinity similar to that displayed by the proteins of the same species. Solution of a binding equation which assumes identical, independent sites, indicated that the number of binding sites on prealbumin for retinol-binding protein is somewhat less than 2 with the human system, and in the neighborhood of 4 with the chicken system. A possible interpretation suggests that prealbumin possesses four identical binding sites for retinol-binding protein, one for each subunit, but that the binding is of a negative cooperative nature. A major share of the negative cooperativity is likely to result from steric hindrance induced by already bound retinol-binding protein molecules, which have a sizable volume compared to the volume of the prealbumin molecule. The cooperativity is likely to be more pronounced with the human system. Rotational relaxation times derived from Perrin plots suggest that 1:1 molecular complexes of retinol-binding proteins with prealbumins have a compact structure.

Self-Assembly of Bovine β-Casein below the Isoelectric pH

Beta-casein is an intrinsically unstructured amphiphilic protein that self-assembles into micelles at neutral pH. This paper reports that beta-casein self-organizes into micelles also under acidic conditions. The protein association behavior and micelle characteristics at pH 2.6, well below the p I, are presented. The pH was found to strongly affect the micelle shape and dimensions. Cryogenic transmission electron microscopy (cryo-TEM) experiments revealed disk-like micelles of 20-25 nm in length and approximately 3.5 nm in height in acidic conditions. An aggregation number of 6 was determined by sedimentation equilibrium under these conditions. Isothermal titration calorimetry experiments verified the association below the p I and allowed determination of the micellization enthalpy, the critical micellar concentration, and the micellization relative cooperativity (MR). Small-angle X-ray scattering results at concentrations below the critical micellization concentration (CMC) suggest that the monomeric protein is likely in a premolten globule state at low pH. Calculations of the protein charge at acidic and neutral pH reveal a similar high net charge but considerable differences in the charge distribution along the protein backbone. Overall the results show that beta-casein is amphiphilic at low pH, but the distribution of charge along the protein chain creates packing constraints that affect the micelle organization, leading at concentrations above the CMC to the formation of disk micelles.

Bio-imprinting of lipases with fatty acids

Bio-imprinting of lipases with fatty acids was shown to be a feasible, effective method for obtaining highly active enzymes in organic solvents. The increase in activity was dependent on the enzyme type, the solvent type and the imprint molecule itself. A correlation between the initial activity of caprylic acid-imprinted Candida rugosa lipase (CRL), and solvent hydrophobicity was observed. In addition, the combination of bio-imprinting with adsorption onto an inert support such as celite, proved to be a powerful technique for obtaining an even more active and stable enzyme preparations. In the case of lipase from Pseudomonas sp., the increase in activity resulting from bio-imprinting with caprylic acid and immobilization onto celite, was 20-fold. Porcine pancreatic lipase (PPL), treated in the same manner, retained 70% of its initial activity at the end of 20 consecutive reaction cycles, compared to only 20% residual activity for the non-treated control.

Dietary oxidized oil enhances the activity of (Na+K+) ATPase and acetylcholinesterase and lowers the fluidity of rat erythrocyte membrane

Abstract The effect of dietary oxidized lipids on the fluidity and function of red blood cell (RBC) membranes was studied. Male growing rats were fed diets containing 10% fresh (control) or oxidized (experimental) soybean oil for 8 weeks. Ingestion of the oxidized oil resulted in the accumulation of high levels of fluorescent peroxidation products in the RBC of the experimental animals. Membrane fluidity was studied by fluorescence polarization, using 1,6-diphenyl-1,3,5 hexatriene (DPH) as a probe. The fluidity of RBC membranes isolated from the animals of the experimental group was significantly lower than that of the membranes derived from the control following 3 and 7 weeks of feeding. This lower fluidity was accompanied by a decrease in the content of membrane polyunsaturated fatty acids and an increase in the activity of the membrane-bound enzymes acetylcholinesterase and (Na + K + )ATPase. Despite the excessive oxidative state of the oil used, the changes observed in membrane composition, dynamics, and function emphasize the potential risk of dietary oxidized lipids.

Subtle differences in structural transitions between poly-L- and poly-D-amino acids of equal length in water

Mirror-image asymmetric molecules, i.e., chiral isomers or enantiomers, are classically considered as chemically identical. Recent studies, however, have indicated that parity violation by the nuclear weak force induces a tiny energy difference between chiral isomers. Upon combination with a massive amplification process, expansion of this difference to a detectable macroscopic level may be achieved. Yet, experimental tests of this possibility, where one enantiomer is compared to the other in solution, are hampered by the possible presence of undetectable impurities. In this study we have overcome this problem by comparing structural and dynamic features of synthetic D- and L-polyglutamic acid and polylysine molecules each of 24 identical residues. In these water-soluble polypeptides helix formation is an intramolecular autocatalytic process amplified by each turn, which is actually unaffected by low level of putative impurities in the solvent. The helix and random coil configurations and their transition were determined in this study by circular dichroism (CD) and isothermal titration calorimetry (ITC) in water and deuterium oxide. Distinct differences in structure and transition energies between the enantiomeric polypeptides were detected by both CD and ITC when dissolved in water. Intriguingly, these differences were by and large abolished in deuterium oxide. Our findings suggest that deviation from physical invariance between the D- and L-polyamino acids is induced in part by different hydration in water which is eliminated in deuterium oxide. Based on the recent findings by Tikhonov and Volkov (V. I. Tikhonov and A. A. Volkov, Science 2002, 296, 2363) we suggest that ortho-H(2)O, which constitutes 75% of bulk H(2)O, has a preferential affinity to L-enantiomers. Differential hydration of enantiomers may have played a role in the selection of L-amino acids by early forms of life.

Dietary oxidized oil and the activity of antioxidant enzymes and lipoprotein peroxidation in rats

The relationship between dietary oxidized oil and the activity of some antioxidant enzymes and the peroxidation state of plasma lipoproteins was studied with male growing rats. The animals were fed diets containing 10% fresh or oxidized soybean oil. Ingestion of the oxidized oil resulted in an elevation in the activity of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase in erythrocytes derived from the experimental rats compared to the controls. In contrast, in animals subjected to the oxidized oil, the hepatic activity of glucose-6-phosphate dehydrogenase was significantly lower, that of glutathione peroxidase and glutathione reductase showed a tendency to decrease, whereas the level of superoxide dismutase activity was unchanged. This could be explained by possible liver damage caused by the oxidized lipids, and indeed, the increased plasma levels of aspartate aminotransferase and alanine aminotransferase activity support this conclusion. Dietary oxidized oil increased the level of lipid peroxidation in various lipoprotein fractions. The study emphasizes the potential risk of dietary oxidized lipids.

Influence of dietary vitamin E and selenium on muscle fatty acid composition in pigs

Abstract A total of 28 female pigs were fed a basal diet containing a low amount of α-tocopherol (10.3 mg/kg; control), and diets supplemented with 0.3 mg selenium/kg (group Se) or with 200 mg α-tocopherol/kg (group V) at the growing-finishing period. Increasing dietary level of vitamin E resulted in higher concentration of α-tocopherol in plasma and muscle immediately after slaughter and 4 h later (P⩽0.05). The fatty acid composition of muscle microsomes and mitochondria was slightly affected by the diet. Corresponding to the minor changes of the membrane fatty acid composition the fluidity was unaffected by the diet. A positive relationship was observed between the resistance to in vitro stimulation of peroxidation and the vitamin E content of the muscle samples. Supplementation with selenium did not reduce the lipid oxidation after stimulation. In conclusion, even though the effect was minor, vitamin E improved the antioxidative status in pork.

Food restriction and membrane fluidity

The fluidity of the erythrocyte membrane derived from growing rats raised under restricted food intake was found to be higher than the fluidity of the respective membranes from ad libitum fed animals. Considering the apparent relationship between the decrease in membrane fluidity and aging, the results point to the possible beneficial effects of food restriction at a young age.