Dynamics and determinants of human plasma bile acid profiles during dietary challenges.

In recent years, bile acids (BA) have received great interest due to their pleiotropic biological activity and the presence of plasma membrane-bound and nuclear receptors. Moreover, BA in blood have been identified by metabolite screening approaches as biomarkers that are associated with various diseases and even with a human longevity phenotype. With the growing interest in the microbiota contribution to the health-disease trajectory, BA that undergo deconjugation and other modifications by bacteria in the large intestine have become a prime target as a microbiome diversity modifier. We here profiled BA by a quantitative and a semiquantitative approach in 15 healthy and phenotypically very similar young individuals for over a 36-h fasting period, an oral glucose tolerance test (OGTT), and an oral lipid tolerance test (OLTT). We demonstrate a remarkable heterogeneity of the responses and describe the different dynamics of the plasma changes that likely originate from different routes by which BA enters the peripheral blood, and that may represent a direct secretion from the liver into the blood and a route that reaches the blood as a spill-over after passing from the gallbladder through the intestine and the portal system. We discuss the finding that an individual transport process involved in the passage of BA could be a critical determinant in the kinetics of plasma appearance and the overall phenotypic variability found.

Myotube Protein Content Associates with Intracellular L-Glutamine Levels.

BACKGROUND/AIMS: Skeletal mass loss is reported in several catabolic conditions and it has been associated with a reduced intracellular L-glutamine content. We investigated the association of intracellular L-glutamine concentration with the protein content in skeletal muscle cells. METHODS: We cultivated C2C12 myotubes in the absence or presence of 2 (reference condition), 8 or 16 mM L-glutamine for 48 hours, and the variations in the contents of amino acids and proteins measured. We used an inhibitor of L-glutamine synthesis (L-methionine sulfoximine - MSO) to promote a further reduction in intracellular L-glutamine levels. Amino acids contents in cells and media were measured using LC-MS/MS. We measured changes in phosphorylated Akt, RP-S6, and 4E-BP1contents in the absence or presence of insulin by western blotting. RESULTS: Reduced intracellular L-glutamine concentration was associated with decreased protein content and increased protein breakdown. Low intracellular glutamine levels were also associated with decreased p-Akt contents in the presence of insulin. A further decrease in intracellular L-glutamine caused by glutamine synthetase inhibitor reduced protein content and levels of amino acids generated from glutamine metabolism and increased bAib still further. Cells exposed to high medium glutamine levels did not have any change in protein content but exhibited increased contents of the amino acids derived from L-glutamine metabolism. CONCLUSION: Intracellular L-glutamine levels per se play a role in the control of protein content in skeletal muscle myotubes.

Roux-en-Y Gastric Bypass Surgery Induces Distinct but Frequently Transient Effects on Acylcarnitine, Bile Acid and Phospholipid Levels.

Roux-en-Y gastric bypass (RYGB) is an effective method to achieve sustained weight loss, but the mechanisms responsible for RYGB effects have not yet been fully characterized. In this study, we profiled the concentrations of 143 lipid metabolites in dry blood spots (DBS) of RYGB patients. DBS from obese patients (BMI range 35⁻44 kg/m²) were collected 7 days before, 15 and 90 days after the surgery. LC-MS/MS was used to quantify acylcarnitines, phosphatidylcholines, sphingomyelins and bile acids. RYGB caused a rapid increase in acylcarnitine levels that proved to be only transient, contrasting with the sustained decrease in phosphatidylcholines and increase of sphingomyelins and bile acids. A PLS-DA analysis revealed a 3-component model (R² = 0.9, Q² = 0.74) with key metabolites responsible for the overall metabolite differences. These included the BCAA-derived acylcarnitines and sphingomyelins with 16 and 18 carbons. We found important correlations between the levels of BCAA-derived acylcarnitines and specific sphingomyelins with plasma cholesterol and triacylglycerol concentrations. Along with the marked weight loss and clinical improvements, RYGB induced specific alterations in plasma acylcarnitines, bile acid and phospholipid levels. This calls for more studies on RYGB effects aiming to elucidate the metabolic adaptations that follow this procedure.

Orange juice affects acylcarnitine metabolism in healthy volunteers as revealed by a mass-spectrometry based metabolomics approach.

Citrus juices, especially orange juice, constitute rich sources of bioactive compounds with a wide range of health-promoting activities. Data from epidemiological and in vitro studies suggest that orange juice (OJ) may have a positive impact on lipid metabolism. However, the effect of orange juice intake on blood lipid profile is still poorly understood. We have used two different blood samples, Dried Blood Spots (DBS) and plasma, to assess the effect of two-week orange juice consumption in healthy volunteers by a mass-spectrometry based metabolomics approach. DBS were analysed by liquid chromatography mass spectrometry (LC-MS) and plasma samples were analysed by the gas chromatography mass spectrometry (GC-MS). One hundred sixty-nine lipids including acylcarnitines (AC), lysophosphatidylcholines (LysoPC), (diacyl- and acyl-alkyl-) phosphatidylcholines (PC aa and PC ae) and sphingomyelins (SM) were identified and quantified in DBS. Eighteen fatty acids were identified and quantified in plasma. Multivariate analysis allowed to identify an increase in C3:1, C5-DC(C6-OH), C5-M-DC, C5:1-DC, C8, C12-DC, lysoPC18:3, myristic acid, pentadecanoic acid, palmitoleic and palmitic acid and a decrease in nervonic acid, C0, C2, C10, C10:1, C16:1, C16-OH, C16:1-OH, C18-OH, PC aa C40:4, PC ae C38:4, PC ae C42:3, PC ae C42:4 and cholesterol levels after orange juice intake. A two-week period of orange juice intake could affect fatty acids ß-oxidation through mitochondrial and peroxisomal pathways, leading to an increase of short-chain acylcarnitines and a decrease of medium and long-chain acylcarnitines. This is the first report analyzing the effect of orange juice intake in healthy volunteers using a dried blood spot-based metabolomics approach.

Medium-chain dicarboxylic acylcarnitines as markers of n-3 PUFA-induced peroxisomal oxidation of fatty acids.

SCOPE: Omega-3 polyunsaturated fatty acids (n-3 PUFA) found in fish oil activate PPAR-α, stimulate peroxisomal fatty acid (FA) ß-oxidation and prevent impairments on glucose homeostasis. METHODS AND RESULTS: Glucose metabolism and FA oxidation were studied in C57/Bl6 mice fed with diets containing either 3.6 and 31.5% fish oil or lard. To assess the effects of peroxisomal proliferation on FA oxidation independent of n-3 PUFA intake, mice were treated with the PPAR-α agonist WY-14643. n-3 PUFA-fed mice were protected from glucose intolerance and dyslipidemia compared to animals fed a lard-based high-fat diet. Most importantly, mice fed on the hyperlipidic diet based on fish oil as well as the WY-14643 treated mice showed twofold increase of odd, medium-chain, dicarboxylic acylcarnitines in the liver suggesting that not only ß-oxidation, but also α- and ω-oxidation of FA were increased. Finally, an oxidation assay using liver homogenates and palmitic acid as substrate revealed an over tenfold increased production of similar acylcarnitines, indicating that FA are their precursors. CONCLUSION: This study shows at the metabolite level that peroxisome proliferation induced either by fish oil or WY-14643 is associated with increased α- and ω-oxidation of FA producing specific acylcarnitines that can be utilized as biomarkers of peroxisomal FA oxidation.

Study of the mechanisms of uptake of 5-aminolevulinic acid derivatives by PEPT1 and PEPT2 transporters as a tool to improve photodynamic therapy of tumours.

Endogenous porphyrin accumulation after administration of 5-aminolevulinic acid is employed in photodynamic therapy of tumours. Due to its low membrane permeability, esterified 5-aminolevulinic acid derivatives less hydrophilic than the parental compound are under investigation. Knowledge of the mechanisms of 5-aminolevulinic acid derivatives uptake into target cells is essential to understand and improve photodynamic therapy and useful in the design of new derivatives with better affinity and with higher selectivity for tumour cells in specific tissues. The aim of this work was to assess the interaction of 5-aminolevulinic acid derivatives with the intestinal PEPT1 and renal transporter PEPT2 expressed in Pichia pastoris yeasts. We found that Undecanoyl, Hexyl, Methyl and 2-(hydroxymethyl)tetrahydropyranyl 5-aminolevulinic acid esters and the dendron 3m-ALA inhibited (14)C-5-aminolevulinic acid uptake by PEPT2. However, only the Undecanoyl ester inhibited 5-aminolevulinic acid uptake by PEPT1. We have also found through a new developed colorimetric method, that Hexyl and 2-(hydroxymethyl)tetrahydropyranyl 5-aminolevulinic acid esters display more affinity than 5-aminolevulinic acid for PEPT2 whereas none of the compounds surpass 5-aminolevulinic acid affinity for PEPT1. In addition, the Undecanoyl ester binds with high affinity to the membranes of PEPT2 and PEPT1-expressing yeasts and to the control yeasts. The main finding of this work was that some derivatives have the potential to improve 5-aminolevulinic acid-based photodynamic therapy by increased efficiency of transport into cells expressing PEPT2 such as kidney, mammary gland, brain or lung whereas in tissues expressing exclusively PEPT1 the parent 5-aminolevulinic acid remains the compound of choice.