Long-term supplementation with CLA-FFA or CLA-triacylglycerol reduces BFM in healthy overweight adults.

After 12 mo in a randomized, double-blind, placebo-controlled trial of conjugated linoleic acid (CLA) supplementation (2 groups received CLA as part of a triglyceride or as the free fatty acid, and 1 group received olive oil as placebo), 134 of the 157 participants who concluded the study were included in an open study for another 12 mo. The goals of the extension study were to evaluate the safety [with clinical chemistry analyses and reported adverse events (AEs)] and assess the effects of CLA on body composition [body fat mass (BFM), lean body mass (LBM), bone mineral mass (BMM)], body weight, and BMI. All subjects were supplemented with 3.4g CLA/d in the triglyceride form. Circulating lipoprotein(a) and thrombocytes increased in all groups. There was no change in fasting blood glucose. Aspartate amino transferase, but not alanine amino transferase, increased significantly. Plasma total cholesterol and LDL cholesterol were reduced, whereas HDL cholesterol and triglycerides were unchanged. The AE rate decreased compared with the first 12 mo of the study. Body weight and BFM were reduced in the subjects administered the placebo during the initial 12 mo study (-1.6 +/- 3.2 and -1.7 +/- 2.8 kg, respectively). No fat or body weight changes occurred in the 2 groups given CLA during the initial 12 mo. LBM and BMM were not affected in any of the groups. Changes in body composition were not related to diet and/or training. In conclusion, this study shows that CLA supplementation for 24 mo in healthy, overweight adults was well tolerated. It confirms also that CLA decreases BFM in overweight humans, and may help maintain initial reductions in BFM and weight in the long term.

Long-term supplementation with conjugated linoleic acid (CLA) reduces body fat mass (BFM) and increases or maintains lean body mass (LBM). However, the regional effect of CLA was not studied. The study aimed to evaluate the effect of CLA per region and safety in healthy, overweight and obese adults. A total of 118 subjects (BMI: 28-32 kg/m 2 ) were included in a double blind, placebo-controlled trial. Subjects were randomised into two groups supplemented with either 3·4 g/d CLA or placebo for 6 months. CLA significantly decreased BFM at month 3 (D ¼ 20·9 %, P¼ 0·016) and at month 6 (D ¼ 2 3·4 %, P¼ 0·043) compared with placebo. The reduction in fat mass was located mostly in the legs (D ¼ 2 0·8 kg, P, 0·001), and in women (D ¼ 21·3 kg, P¼ 0·046) with BMI . 30 kg/m 2 (D ¼ 21·9 kg, P¼ 0·011), compared with placebo. The waist -hip ratio decreased significantly (P¼ 0·043) compared with placebo. LBM increased (D ¼ þ0·5 kg, P¼ 0·049) within the CLA group. Bone mineral content was not affected (P¼ 0·70). All changes were independent of diet and physical exercise. Safety parameters including blood lipids, inflammatory and diabetogenic markers remained within the normal range. Adverse events did not differ between the groups. It is concluded that supplementation with CLA in healthy, overweight and obese adults decreases BFM in specific regions and is well tolerated.

FYVE zinc finger domains, which are conserved in multiple proteins from yeast to man, interact specifically with the membrane lipid phosphatidylinositol 3-phosphate (PtdIns(3)P). Here we have investigated the structural requirements for the interaction of the FYVE finger of the early endosome antigen EEA1 with PtdIns(3)P and early endosomes. The binding of the FYVE finger to PtdIns(3)P is Zn 2؉ -dependent, and Zn 2؉could not be replaced by any other bivalent cations tested. By surface plasmon resonance, the wild-type FYVE finger was found to bind to PtdIns(3)P with an apparent K D of about 50 nM and a 1:1 stoichiometry. Mutagenesis of cysteines involved in Zn 2؉ coordination, basic residues thought to be directly involved in ligand binding and other conserved residues, resulted in a 6-to >100-fold decreased affinity for PtdIns(3)P. A mutation in the putative PtdIns(3)P-binding pocket, R1375A, may prove particularly informative, because it led to a strongly decreased affinity for PtdIns(3)P without affecting the FYVE three-dimensional structure, as measured by fluorescence spectroscopy. Whereas the C terminus of EEA1 localizes to early endosomes when expressed in mammalian cells, all the FYVE mutants with reduced affinity for PtdIns(3)P were found to be largely cytosolic. Furthermore, whereas expression of the wild-type EEA1 C terminus interferes with early endosome morphology, the point mutants were without detectable effect. These results support recently proposed models for the ligand binding of the FYVE domain and indicate that PtdIns(3)P binding is crucial for the localization and function of EEA1.Phosphatidylinositol 3-kinases are important regulators of vital cellular processes, including endocytic membrane trafficking, signal transduction, apoptosis, and cytoskeletal organization (1-3). The recent discovery of the conserved FYVE zinc finger (for Fab1p, YOTB, Vac1p, EEA1) 1 (4) as a phosphatidylinositol 3-phosphate (PtdIns (3)P) -specific domain has shed light on the function of this phosphatidylinositol 3-kinase product in cellular processes (5-8). FYVE finger proteins include the membrane trafficking regulators EEA1, Hrs, Vac1p, Vps27p, and Fab1p, the signal transducer SARA, the putative cytoskeletal regulator Fgd1, as well as a number of proteins with unknown function (5, 9 -15).The determination of the crystal structure of the ligand-free FYVE domain of the yeast vacuolar sorting protein Vps27p at high resolution has enabled the modeling of the FYVE-PtdIns (3)P interaction (16). According to this model, the basic residues present in the ␤1 strand ( Fig.

Neutral red is a lysosomal probe and a biological pH indicator. In aqueous solutions, the protonated (NRH) and neutral (NR) forms of monomeric neutral red exhibit distinct absorption maxima (535 and 450 nm, respectively) but have the same fluorescence with a maximum at 637 nm and a quantum yield of 0.02. The similarity of the fluorescence spectra at acidic and basic pH suggests deprotonation of cationic species in the first singlet excited state. The NR fluorescence strongly depends on the solvent polarity as shown by addition of increasing amounts of water to pure dioxane, which gradually shifts the fluorescence maximum from 540 nm in pure dioxane to 637 nm in water. The fluorescence quantum yield increases from 0.17 in dioxane to 0.3 upon addition of 7% water and then decreases, reaching 0.02 in pure water. Immediately after incubation of human skin fibroblasts with neutral red, excitation with 435 nm light produces a fluorescence whose maximum is recorded at 575 nm. This fluorescence is located in the perinuclear region and originates from large fluorescent intracytoplasmic spots, suggesting staining of the endoplasmic reticulum-Golgi complex. At longer times, this fluorescence is shifted to 606 nm, suggesting slow diffusion of the lysosomotropic dye toward the more hydrated and acidic interior of lysosomes. Addition of a lysosomotropic detergent to cells previously incubated with neutral red shifts the fluorescence to the blue. Thus, in complex biological systems, this probe cannot be a good pH indicator but is a very sensitive probe of lysosomal microenvironments.

The subcellular localization of protoporphyrin (PP) has been studied by microspectrofluorometric techniques in NCTC 2544 keratinocytes incubated with 5-aminolevulinic acid (ALA) for times up to 42 h. Whereas the plasma membrane shows strong staining, fluorescent spots are observed within the cytoplasm especially in the perinuclear region. Although the topographic pattern of the PP distribution does not change much with the incubation time with ALA, the fluorescence spectra suggest that the PP microenvironments are quite different at short and long incubation times. Addition of 18 microM desferrioxamine almost doubles the ALA-induced PP concentration. Colocalization experiments with rhodamine 123, a mitochondrial probe, and lucifer yellow (LY) or neutral red (NR), two lysosome probes, demonstrate that at least some of these spots are of lysosomal origin. Study of the time evolution of the NR fluorescence under irradiation with visible light in the presence and absence of ALA demonstrates that lysosomes are damaged cells that have synthesized PP. No PP fluorescence can be detected in mitochondria after incubation with ALA. However, photosensitization of mitochondria occurs under irradiation with visible light. Very little formation of lipofuscins by photosensitization with exogenous PP or ALA-induced PP is observed with the NCTC 2544 keratinocytes, as compared to normal human fibroblasts.

CLA mixtures are now commercially available. They differ from each other with respect to their content of CLA isomers and their degree of purification. As a group of natural FA, CLA have been widely assumed to be safe. However, the suspected presence of both impurities and particular isomers might induce undesirable side effects. Despite this potential health risk, only a few CLA preparations have been tested under rigorous conditions for clinical efficacy and safety. Based on the limited results available, it is possible to suggest that preparations enriched in c9,t11 and t10,c12 isomers are preferable for human consumption compared to preparations containing four isomers, in terms both of safety and efficacy.

Lentinus edodes (Shiitake) is a medicinal mushroom with a long tradition of use in Asia. The major active substance in L. edodes is a (1-6,1-3)-beta-glucan (lentinan). No clinical controlled studies have yet investigated the effect of orally administered lentinan on the immune response in healthy, elderly Caucasian subjects. We evaluated the effect and the safety of a beta-glucan from L. edodes mycelium, Lentinex, in healthy, elderly subjects in a double blind, crossover, placebo-controlled trial. Forty-two subjects were randomly allocated to two groups given orally either 2.5 mg/day Lentinex or placebo for 6 weeks; then after a washout period of 4 weeks, the alternate supplementation was given for 6 weeks. The changes in the number of B-cells were significantly different between the groups. The number ofNK cells increased significantly in both groups, but there was no significant difference between the groups. Other factors of the immune response (immunoglobulins, complement proteins, cytokines) were not altered. The safety blood variables (differential cell count, liver function, kidney function, and other blood chemistry) were not influenced by Lentinex, and the number, nature, and severity of adverse events were similar to placebo. Lentinex given orally to elderly subjects was safe and induced an increase in the number of circulating B-cells.