Short communication: Effect of supplementation with Lactobacillus casei Shirota on insulin sensitivity, ß-cell function, and markers of endothelial function and inflammation in subjects with metabolic syndrome--a pilot study.

Based on animal studies, intake of probiotic bacteria was suggested to improve insulin sensitivity by reducing endotoxinemia and inflammation. The objective of this study was to determine the effects of supplementation with the probiotic strain Lactobacillus casei Shirota (LcS) over 12 wk on insulin sensitivity, ß-cell function, inflammation, and endothelial dysfunction parameters in subjects with metabolic syndrome. In a randomized-controlled study, 30 subjects with metabolic syndrome either received Lactobacillus casei Shirota 3 times daily for 12 wk or served as controls with standard medical therapy. Fasting blood samples were taken and a 75-g oral glucose tolerance test was performed to derive indices for insulin sensitivity and ß-cell function. In addition, parameters to assess endothelial function and inflammation markers were determined. Even though the insulin sensitivity index significantly improved after 3 mo of probiotic supplementation (0.058±0.021 vs. 0.038±0.025), the change was not significantly different compared with the control group. No improvements were seen in additional indices of insulin sensitivity (quantitative insulin sensitivity check index, insulin sensitivity by oral glucose tolerance test, and homeostasis model assessment for insulin resistance) and ß-cell function (first and second phase insulin secretion, and homeostasis model assessment for ß-cell function). Probiotic supplementation resulted in a significant reduction in soluble vascular cell adhesion molecule-1 (sVCAM-1) level (1,614±343 vs. 1,418±265 ng/mL). No significant changes in parameters used to assess low-grade inflammation or endothelial dysfunction were observed. Intake of LcS for 12 wk in subjects with metabolic syndrome did not improve insulin sensitivity, ß-cell function, endothelial function, or inflammation markers in this trial.

Cellular and plasma antioxidant activity assay using tetramethoxy azobismethylene quinone.

The kinetics of the reduction of enzymatically generated tetramethoxy azobismethylene quinone (TMAMQ), a newly developed antioxidant activity assay method, by pure cellular and plasma antioxidants was studied. Further, the potential application of TMAMQ to the estimation of the antioxidant activity of clinical serum samples was investigated. The highest reduction rate (k) was obtained with ascorbic acid (1.11x10(-2)microM(-1) s(-1)) and glutathione showed the lowest (2.94x10(-5)microM(-1) s(-1)). Comparing TMAMQ and the commercially available antioxidant method Total Antioxidant Capacity clearly shows a similar trend, although the values differ. This study also shows that TMAMQ is highly sensitive (only a minute plasma sample was required) and reproducible, and the reaction proceeds until steady state (until all antioxidants have reacted). TMAMQ is very stable in acetonitrile (>3months), making it a highly flexible method because it can be easily adapted for analysis of just a single sample or for high-throughput analysis. This has direct implications on reducing costs and experimental steps. TMAMQ is therefore a highly promising antioxidant activity assay method for cellular and plasma antioxidant activity assay.

Competitive and seasonal oxidative stress in elite alpine ski racers.

We investigated competitive- and long-term oxidative stress during a competition season in eight top-ranked members of the Austrian Men's Alpine Ski Team. Serum total peroxides, antibody titers against oxidized LDL (oLAb) and lag time of the degradation of the fluorophore 1-palmitoyl-2-((2-(4-(6-phenyl-trans-1,3,5-hexatrienyl)phenyl)ethyl)-carbonyl)-sn-glycero-3-phosphocholine were measured, along with plasma concentrations of ascorbate, alpha- and gamma-tocopherol, beta-carotene, uric acid and the lipid status. Competitive stress was indicated through an increased post-race uric acid level (286 +/- 50 microM pre-race vs 456 +/- 77 microM post-race, P<0.001) in December. Long-term effects were already apparent in November, with the highest concentrations of total peroxides (680 +/- 458 microM H(2)O(2) equivalents vs December 47 +/- 58 microM H(2)O(2) equivalents and January 15 +/- 28 microM H(2)O(2) equivalents, P<0.001) and a concomitant decrease in oLAb titers with an antibody trough in December (439 +/- 150 mU/mL vs baseline 1036 +/- 328 mU/mL; P=0.003). In January, after recovery, they attained nearly pre-season levels of oxidative stress biomarkers. This study indicates midseason oxidative stress in top-level skiers, which was associated with the performance in these athletes.

Lipid peroxidation and antioxidant status in professional American football players during competition.

BACKGROUND: Oxidative stress occurs during strenuous physical exercise, perhaps as a result of increased consumption of oxygen. MATERIALS AND METHODS: In this study, different markers of oxidative stress were determined in eight national league American football players. Before (March) and at three time-points during the competition season (May, June, July) serum total peroxide concentrations, auto-antibody titres against oxidized low-density lipoprotein (oLab), and lag time of reactive oxygen species-induced degradation of the fluorophore 1-palmitoyl-2-((2-(4-(6-phenyl-trans-1,3,5-hexatrienyl)phenyl)ethyl)- carbonyl)-sn-glycero-3-phosphocholine (DPHPC) were measured along with serum ascorbate, alpha- and gamma-tocopherol, and beta-carotene concentrations. RESULTS: Before the competition season, serum antioxidant concentrations were within the lower normal range; ascorbate concentrations increased significantly during the competition period (P < 0.05). Serum peroxide concentrations were within the normal range and increased significantly during the competition period (P < 0.05); in four of the eight subjects the increase was several times the baseline values, while four athletes did not show any increase. The oLab titres increased significantly at the mid-competition period time-point (P < 0.01), but levelled off thereafter. DISCUSSION: Given that it could not be predicted from the baseline oxidative stress and antioxidant status which subject would respond to strenuous exercise with an increase in oxidative stress status, it is concluded that oxidative stress should be monitored in all athletes.

Relationship between classic risk factors, plasma antioxidants and indicators of oxidant stress in angina pectoris (AP) in Tehran.

Cardiovascular disease (CVD) in general seems to be the leading cause of death in the Eastern Mediterranean Region (EMR) including Iran. This may be due to classic risk factors such as high triglyceride (TG), high total cholesterol (TC), and low levels of high density lipoprotein cholesterol (HDL-C). The impact of antioxidants as potentially protective risk factors against early coronary heart disease (CHD) is unknown in Iran. Therefore, relationships between angina and plasma antioxidants and indicators of lipid peroxidation were investigated in a case-control study. In this study, 82 cases of previously undiagnosed angina pectoris (AP), identified by a modified WHO Rose chest pain questionnaire and verified by electrocardiography during treadmill exercise testing, were compared with 146 controls selected from the same population of over 4000 male civil servants aged 40-60 years. Subjects with AP declared significantly less physical activity and had higher serum TG [means (S.E.M.) 2.32 (0.18) versus 1.61 (0.07) mmol/l] but lower HDL-C [1.01 (0.04) versus 1.18 (0.03) mmol/l] than age-matched controls. Levels of total serum cholesterol, low-density lipoprotein cholesterol (LDL-C) and lipoprotein(a) [Lp(a)] were not significantly different between the two groups, while the ratio of LDL-C/HDL-C was significantly higher [4.51 (0.23) versus 3.54 (0. 11)] for subjects with AP than for the controls. There was no significant difference in plasma levels of alpha-tocopherol, vitamin C, alpha- and beta-carotene. However, retinol [1.90 (0.06) versus 2. 09 (0.05)] and beta-cryptoxanthin [0.398 (0.04) versus 0.467 (0.03)] were significantly lower in AP. Furthermore, angina cases exhibited a higher index of lipid peroxidation than controls (e.g. malondialdehyde, MDA; 0.376 (0.010) versus 0.337 (0.009) micromol/l). On multiple logistic regression analysis, retinol with odds ratio (OR) of 0.644 [95% confidence interval (CI; 0.425-0.978)], beta-cryptoxanthin, with an OR of 0.675 (CI; 0.487-0.940), oxidation indices, MDA with OR of 1.612 (95% CI; 1.119-2.322) and LDL-C/HDL-C ratio with OR of 2.006 (95% CI; 1.416-2.849) showed the most significant independent associations with AP in this group of Iranians. In conclusion, the state of lipid peroxidation as well as the status of special antioxidants may be co-determinants of AP in Iran, in parallel with the influence of classical risk factors for cardiovascular disease.

Effect of iodide on total antioxidant status of human serum.

Free radicals and subsequent lipid peroxidation have been implicated in the pathogenesis of several degenerative and chronic diseases which are also treated frequently in spas. There are some data arising from previous studies which support an antioxidant or scavenging effect of iodide, being the essential ingredient of a therapeutically used local brine. The aim of the study was to test the antioxidant capacity of iodide in human serum. For this reason we measured the so-called Total Antioxidant Status determined by a colorimetric method, which reflects the protection against the attack of reactive oxygen species, including enzymic and non-enzymic antioxidants. Exogenous iodide applied as NaI, shows a significantly increased antioxidant capacity in comparison with NaCl at a concentration of 15 microM, which is quite comparable to the upper range of serum iodide levels achieved through balneo-therapeutical intervention. This result is in accordance with previous results from in vitro depolymerization experiments with hyaluronic acid. The antioxidant effect of 15 microM NaI has been found to be approaching the physiologically relevant concentration of ascorbic acid (50 microM).

Treatment of the budding yeast Saccharomyces cerevisiae with the lipid peroxidation product 4-HNE provokes a temporary cell cycle arrest in G1 phase.

The effects of 4-hydroxy-2-nonenal (HNE) on the cell division cycle were investigated in the yeast Saccharomyces cerevisiae. A short treatment with HNE at a concentration in the range of the IC50 value in S. cerevisiae SP-4 cells induced a significant increase in the proportion of G0/G1 cells at the expense of S-phase cells. A similar delay in cell cycle progression upon treatment with HNE has recently been shown for HL-60 neoplastic cells. Long-term exposure in a synchronized yeast culture resulted in a pronounced dose-dependent block between G0G1- and S-phase, probably at or close to a point in the cell cycle that has been designated as "START." Incorporation of radioactively labeled precursors of macromolecules revealed that DNA synthesis was most susceptible to HNE in comparison to RNA and protein synthesis. Production of glutathione appeared to be required for the continuation of the cell cycle. HNE-treated yeast cells reentered the cell cycle when their glutathione content exceeded about twice the level of control cells. The release from the cell division cycle delay was followed by an enhanced growth to an extent that HNE-treated cells exceeded the number of control cells. These results indicate that HNE causes a biphasic modulation of cell proliferation. It was concluded that this effect was conserved during evolution from yeast to mammalian cells, emphasizing once more the usefulness of this unicellular organism as a model system for the investigation of the effects of free radical-derived products on the proliferation of eukaryotes.

Increased stress parameter synthesis in the yeast Saccharomyces cerevisiae after treatment with 4-hydroxy-2-nonenal.

The metabolism of glutathione (GSH), a marker of oxidative stress and trehalose, a rather general physiological stress marker, was examined in exponentially growing Saccharomyces cerevisiae cells after treatment with 4-hydroxynonenal (HNE). GSH was entirely depleted within a 2 h incubation with 250 microM HNE. After removal of the aldehyde it was replenished by de novo synthesis leading to an overshooting GSH level, which later decreased to the basal level. In addition, trehalose was elevated 4-fold in HNE-treated yeast cells compared to control cells. We conclude that increased GSH levels upon HNE treatment are a general phenomenon of eukaryotic cells to ensure protection and survival during further harsh conditions. Furthermore, we have discovered a new indication for the stress marker trehalose in S. cerevisiae.

Quantitative determination of oLAb titers in various animal species.

It is generally accepted, that lipid peroxidation plays a pathogenic role in atherosclerosis. Furthermore, recent studies indicate that antibodies directed against oxidative modifications of Low Density Lipoprotein (oLAb) contribute to atherosclerotic processes and may have some function in other disorders. These antibodies have been determined predominantly in humans, because assays for oLAb measurement use species specific anti IgG conjugates. From such assay designs it is not possible to get directly comparable data from various animal species. Main advantages of comparable data between animal species are that results of animal experiments can be interpreted using human calibrators and that results of immunisations and production of monoclonal antibodies are directly comparable not only within, but also between animal species. The aim of this study was to find a modification for ELISAs for oLAb determination, which allows to measure sera of various animal species simultaneously. Microtitration plates were coated with oxidised LDL and blocked with bovine serum albumine. Human and animal sera were then pipetted into the plate in logarithmic serial dilutions and incubated for 2 h at 37 degrees C. After washing, a protein A horse-radish peroxidase conjugate (Biomakor, Israel) was added to each well in a dilution of 1:20,000. The incubation conditions had to be optimized to achieve reliable results. After another washing step, the assay was developed with TMB. Absorptions were read at 450 nm in a microplate photometer. Following the manufacturers incubation instructions, which recommended a duration of 1 h at room temperature, the system did not work optimally. No binding of protein A to IgG molecules bound to oxidised LDL could be observed, if the system was incubated at 37 degrees C. In our hands, best results were achieved for several animal species, if the conjugate was incubated for two hours at 2-4 degrees C in a refrigerator. Under these conditions, assay sensitivity was the same as in the standard method, which uses anti-species IgG conjugates. The protein A modification of oLAb allows direct reading of animal oLAb titres from human calibrators. With this method, results of animal experiments can be interpreted on the basis of the situation in humans. Preliminary results obtained show that immunisation experiments with oxidised LDL give serum titres in animals, which are in the same order of magnitude as human sera with high oLAb concentrations. The results of this study, in accordance with findings of other authors, give further indications that atherosclerotic processes are influenced by the specific immune system.

Assessment of growth inhibition by aldehydic lipid peroxidation products and related aldehydes by Saccharomyces cerevisiae.

The effect of pretreatment with aldehydes on the subsequent colony forming efficiency (CFE) of Saccharomyces cerevisiae was investigated. All 21 aldehydes tested inhibited CFE in a dose-dependent manner. The effective doses, however, differed markedly from 300 mM to 0.07 mM depending on the functional groups and chain length of the aldehydes. Amongst the nine representatives of n-alkanals, formaldehyde was the most potent inhibitor, reducing CFE to 50 per cent at a dose of 0.3 mM (IC50). In the series of 2-trans-alkenals, acrolein was most effective with an IC50 of 0.08 mM and amongst the 4-hydroxy 2-trans-alkenals, 4-hydroxynonenal was most effective with IC50 of 0.07 mM. In general, effectiveness decreased in the order: 4-hydroxyalkenals > 2-alkenals >> n-alkenals. It is proposed that S. cerevisiae is a promising target cell to elucidate further the molecular mechanisms by which aldehydes, particularly the lipid peroxidation product 4-hydroxynonenal, inhibits cell proliferation.