Evaluation and Characterization of Bacterial Metabolic Dynamics with a Novel Profiling Technique, Real-Time Metabolotyping

BACKGROUND: Environmental processes in ecosystems are dynamically altered by several metabolic responses in microorganisms, including intracellular sensing and pumping, battle for survival, and supply of or competition for nutrients. Notably, intestinal bacteria maintain homeostatic balance in mammals via multiple dynamic biochemical reactions to produce several metabolites from undigested food, and those metabolites exert various effects on mammalian cells in a time-dependent manner. We have established a method for the analysis of bacterial metabolic dynamics in real time and used it in combination with statistical NMR procedures. METHODOLOGY/PRINCIPAL FINDINGS: We developed a novel method called real-time metabolotyping (RT-MT), which performs sequential (1)H-NMR profiling and two-dimensional (2D) (1)H, (13)C-HSQC (heteronuclear single quantum coherence) profiling during bacterial growth in an NMR tube. The profiles were evaluated with such statistical methods as Z-score analysis, principal components analysis, and time series of statistical TOtal Correlation SpectroScopY (TOCSY). In addition, using 2D (1)H, (13)C-HSQC with the stable isotope labeling technique, we observed the metabolic kinetics of specific biochemical reactions based on time-dependent 2D kinetic profiles. Using these methods, we clarified the pathway for linolenic acid hydrogenation by a gastrointestinal bacterium, Butyrivibrio fibrisolvens. We identified trans11, cis13 conjugated linoleic acid as the intermediate of linolenic acid hydrogenation by B. fibrisolvens, based on the results of (13)C-labeling RT-MT experiments. In addition, we showed that the biohydrogenation of polyunsaturated fatty acids serves as a defense mechanism against their toxic effects. CONCLUSIONS: RT-MT is useful for the characterization of beneficial bacterium that shows potential for use as probiotic by producing bioactive compounds

Absorption and Metabolism of cis-9,trans-11-CLA and of Its Oxidation Product 9,11-Furan Fatty Acid by Caco-2 Cells

Furan fatty acids (furan-FA) can be formed by auto-oxidation of conjugated linoleic acids (CLA) and may therefore be ingested when CLA-containing foodstuff is consumed. Due to the presence of a furan ring structure, furan-FA may have toxic properties, however, these substances are toxicologically not well characterized so far. Here we show that 9,11-furan-FA, the oxidation product of the major CLA isomer cis-9,trans-11-CLA (c9,t11-CLA), is not toxic to human intestinal Caco-2 cells up to a level of 100 μM. Oil-Red-O staining indicated that 9,11-furan-FA as well as c9,t11-CLA and linoleic acid are taken up by the cells and stored in the form of triglycerides in lipid droplets. Chemical analysis of total cellular lipids revealed that 9,11-furan-FA is partially elongated probably by the enzymatic activity of cellular fatty acid elongases whereas c9,t11-CLA is partially converted to other isomers such as c9,c11-CLA or t9,t11-CLA. In the case of 9,11-furan-FA, there is no indication for any modification or activation of the furan ring system. From these results, we conclude that 9,11-furan-FA has no properties of toxicological relevance at least for Caco-2 cells which serve as a model for enterocytes of the human small intestine

The effect of conjugated linoleic acid supplementation after weight loss on body weight regain, body composition, and resting metabolic rate in overweight subjects

The effect of conjugated linoleic acid supplementation after weight loss on body weight regain, body composition, and resting metabolic rate in overweight subjects. Kamphuis MM, Lejeune MP, Saris WH, Westerterp-Plantenga MS. Department of Human Biology, Faculty of Health Sciences, Maastricht University, The Netherlands. [email protected] OBJECTIVE: To study the effects of 13 weeks conjugated linoleic acid (CLA) supplementation in overweight subjects after weight loss on weight regain, body composition, resting metabolic rate, substrate oxidation, and blood plasma parameters. DESIGN: This study had a double-blind, placebo-controlled randomized design. Subjects were first submitted to a very-low-calorie diet (VLCD 2.1 MJ/d) for 3 weeks after which they started with the 13-week intervention period. They either received 1.8 g CLA or placebo per day (low dosage, LD) or 3.6 g CLA or placebo per day (high dosage, HD). SUBJECTS: A total of 26 men and 28 women (age 37.8+/-7.7 y; body mass index (BMI) 27.8+/-1.5 kg/m(2)). MEASUREMENTS: Before VLCD (t=-3), after VLCD but before CLA or placebo intervention (t=0) and after 13-week CLA or placebo intervention (t=13), body weight, body composition (hydrodensitometry and deuterium dilution), resting metabolic rate, substrate oxidation, physical activity, and blood plasma parameters (glucose, insulin, triacylglycerol, free fatty acids, glycerol and beta-hydroxy butyrate) were measured. RESULTS: The VLCD significantly lowered body weight (6.9+/-1.7%), %body fat, fat mass, fat-free mass, resting metabolic rate, respiratory quotient and plasma glucose, insulin, and triacylglycerol concentrations, while free fatty acids, glycerol and beta-hydroxy butyrate concentrations were increased. Multiple regression analysis showed that at the end of the 13-week intervention, CLA did not affect %body weight regain (CLA LD 47.9+/-88.2%, CLA HD 27.4+/-29.8%, Placebo LD 32.0+/-42.8%, Placebo HD 22.5+/-37.9%). The regain of fat-free mass was increased by CLA (LD 6.2+/-3.9, HD 4.6+/-2.4%) compared to placebo (LD 2.8+/-3.2%, HD 3.4+/-3.6%), independent of %body weight regain and physical activity. As a consequence of an increased regain of fat-free mass by CLA, resting metabolic rate was increased by CLA (LD 12.0+/-11.4%, HD 13.7+/-14.4%) compared to placebo (LD 9.1+/-11.0%, HD 8.6+/-8.5%). Substrate oxidation and blood plasma parameters were not affected by CLA. CONCLUSION: In conclusion, the regain of fat-free mass was favorably, dose-independently affected by a 13-week consumption of 1.8 or 3.6 g CLA/day and consequently increased the resting metabolic rate. However, it did not result in improved body weight maintenance after weight loss