On the physical-chemical properties of ceramide C16

Dissertation presented to obtain the Ph.D degree in Chemistry.

Comparison of oleate conversion under microaerophilic and anaerobic conditions

Excessive accumulation of Long Chain Fatty Acids (LCFA) in methanogenic bioreactors is the cause of process failure associated to a severe decrease in methane production. In particular, fast and persistent accumulation of palmitate is critical and still not elucidated. Aerobes or facultative anaerobes were detected in those reactors, raising new questions on LCFA biodegradation. To get insight into the influence of oxygen, two bioreactors were operated under microaerophilic and anaerobic conditions, with oleate at 1 and 4 gCOD/(L d). Palmitate accumulated up to 2 and 16 gCOD/L in the anaerobic and microaerophilic reactor, respectively, which shows the importance of oxygen in this conversion. A second experiment was designed to understand the dynamics of oleate to palmitate conversion. A CSTR and a PFR were assembled in series and fed with oleate under microaerophilic conditions. HRT from 6 to 24 h were applied in the CSTR, and 14 to 52 min in the PFR. In the PFR a biofilm was formed where palmitate accounted for 82% of total LCFA. Pseudomonas was the predominant genus (42 %) in this biofilm, highlighting the role of aerobic and facultative anaerobic bacteria in LCFA bioconversion.

Cathepsin D protects colorectal cancer cells from acetate-induced apoptosis through autophagy-independent degradation of damaged mitochondria

Acetate is a short-chain fatty acid secreted by Propionibacteria from the human intestine, known to induce mitochondrial apoptotic death in colorectal cancer (CRC) cells. We previously established that acetate also induces lysosome membrane permeabilization in CRC cells, associated with release of the lysosomal protease cathepsin D (CatD), which has a well-established role in the mitochondrial apoptotic cascade. Unexpectedly, we showed that CatD has an antiapoptotic role in this process, as pepstatin A (a CatD inhibitor) increased acetate-induced apoptosis. These results mimicked our previous data in the yeast system showing that acetic acid activates a mitochondria-dependent apoptosis process associated with vacuolar membrane permeabilization and release of the vacuolar protease Pep4p, ortholog of mammalian CatD. Indeed, this protease was required for cell survival in a manner dependent on its catalytic activity and for efficient mitochondrial degradation independently of autophagy. In this study, we therefore assessed the role of CatD in acetate-induced mitochondrial alterations. We found that, similar to acetic acid in yeast, acetate-induced apoptosis is not associated with autophagy induction in CRC cells. Moreover, inhibition of CatD with small interfering RNA or pepstatin A enhanced apoptosis associated with higher mitochondrial dysfunction and increased mitochondrial mass. This effect seems to be specific, as inhibition of CatB and CatL with E-64d had no effect, nor were these proteases significantly released to the cytosol during acetate-induced apoptosis. Using yeast cells, we further show that the role of Pep4p in mitochondrial degradation depends on its protease activity and is complemented by CatD, indicating that this mechanism is conserved. In summary, the clues provided by the yeast model unveiled a novel CatD function in the degradation of damaged mitochondria when autophagy is impaired, which protects CRC cells from acetate-induced apoptosis. CatD inhibitors could therefore enhance acetate-mediated cancer cell death, presenting a novel strategy for prevention or therapy of CRC.

Effect of sulfate and iron (III) on LCFA degradation by a methanogenic community

[Excerpt] Under anaerobic conditions long chain fatty acids (LCFA) can be converted to methane by syntrophic bacteria and methanogenic archaea. LCFA degradation was also reported in the presence of alternative hydrogenotrophic partners, such as sulfate-reducing bacteria (SRB) and iron-reducing bacteria (IRB), which generally show higher affinity for H2 than methanogens and are more resistant to LCFA [1,2,3]. Their presence in a microbial culture degrading LCFA can be advantageous to reduce LCFA toxicity towards methanogens, although high concentrations of external electron acceptor (EEA) can lead to outcompetition of methanogens and cease methane production. In this work, we tested the effect of adding sub-stoichiometric concentrations of sulfate and iron(III) to methanogenic communities degrading LCFA. (...)

Energy production from lipids by novel anaerobes

[Excerpt] Anaerobic microbial diversity encloses a very high potential that can be used for biotechnological applications. This potential is still largely unexplored, since the majority of the microorganisms in Nature are unknown or poorly characterized. This work is focused on the study of novel anaerobic microorganisms that participate in the metabolism of lipids, long chain fatty acids (LCFA) and glycerol, with the main goal of producing valuable energy-rich organic compounds. For that, conventional anaerobic culturing procedures were combined with continuous bioreactors operation and allied to microbial ecology approaches. Two main examples of the work performed will be presented. (...)

Conversion of Cn-unsaturated into Cn-2-saturated LCFA can occur uncoupled from methanogenesis in anaerobic bioreactors

Fat, oils, and grease present in complex wastewater can be readily converted to methane, but the energy potential of these compounds is not always recyclable, due to incomplete degradation of long chain fatty acids (LCFA) released during lipids hydrolysis. Oleate (C18:1) is generally the dominant LCFA in lipid-containing wastewater, and its conversion in anaerobic bioreactors results in palmitate (C16:0) accumulation. The reason why oleate is continuously converted to palmitate without further degradation via β-oxidation is still unknown. In this work, the influence of methanogenic activity in the initial conversion steps of unsaturated LCFA was studied in 10 bioreactors continuously operated with saturated or unsaturated C16- and C18-LCFA, in the presence or absence of the methanogenic inhibitor bromoethanesulfonate (BrES). Saturated Cn-2-LCFA accumulated both in the presence and absence of BrES during the degradation of unsaturated Cn-LCFA, and represented more than 50\% of total LCFA. In the presence of BrES further conversion of saturated intermediates did not proceed, not even when prolonged batch incubation was applied. As the initial steps of unsaturated LCFA degradation proceed uncoupled from methanogenesis, accumulation of saturated LCFA can be expected. Analysis of the active microbial communities suggests a role for facultative anaerobic bacteria in the initial steps of unsaturated LCFA biodegradation. Understanding this role is now imperative to optimize methane production from LCFA.

Paper de RIP140 en la fisiopatogenia de la adrenoleucodistròfia lligada a l'X.

La adrenoleucodistrofia ligada al X (X-ALD) es un enfermedad neurometabólica fatal caracterizada por una desmielinización cerebral progresiva infantil (CCALD) o por una neurodegeneración de la médula espinal (adrenomieloneuropatía, AMN), insuficiencia adrenal y acumulación de ácidos grasos de cadena muy larga (AGCML) como el ácido hexacosanoico (C26:0) en tejidos. La enfermedad está causada por mutaciones en el gen ABCD1 el cual codifica para un transportador peroxisomoal que importa AGCML. El ratón knockout para Abcd1 (Abcd1-) desarrolla alteraciones en la médula espinal que mimetizan el modelo de enfermedad AMN con inicio de los síntomas a los 20 meses. Previamente, nuestro grupo evidenció mediante análisis de transcriptómica, una desregulación mitocondrial en el modelo murino Abcd1- . En este trabajo demostramos que tanto en el ratón Abcd1- como en la sustancia blanca afectada de pacientes X-ALD hay una depleción mitocondrial. Para poder explicar esta depleción, estudiamos los niveles de un repressor de la biogenesis mitocondrial, RIP140. En cultivo organotípico de cortes de médula espinal observamos un aumento de los niveles proteicos de RIP140 en el ratón Abcd1- y también un aumento mediado por C26:0. Estos resultados indican que la sobreexpresión de RIP140 puede ser la responsable de la depleción mitocondrial presente en el ratón Abcd1- y una posible nueva diana terapèutica para la X-ALD.

ELOVL6 Genetic Variation Is Related to Insulin Sensitivity: A New Candidate Gene in Energy Metabolism

BACKGROUND: The elongase of long chain fatty acids family 6 (ELOVL6) is an enzyme that specifically catalyzes the elongation of saturated and monounsaturated fatty acids with 12, 14 and 16 carbons. ELOVL6 is expressed in lipogenic tissues and it is regulated by sterol regulatory element binding protein 1 (SREBP-1). OBJECTIVE: We investigated whether ELOVL6 genetic variation is associated with insulin sensitivity in a population from southern Spain. DESIGN: We undertook a prospective, population-based study collecting phenotypic, metabolic, nutritional and genetic information. Measurements were made of weight and height and the body mass index (BMI) was calculated. Insulin resistance was measured by homeostasis model assessment. The type of dietary fat was assessed from samples of cooking oil taken from the participants' kitchens and analyzed by gas chromatography. Five SNPs of the ELOVL6 gene were analyzed by SNPlex. RESULTS: Carriers of the minor alleles of the SNPs rs9997926 and rs6824447 had a lower risk of having high HOMA_IR, whereas carriers of the minor allele rs17041272 had a higher risk of being insulin resistant. An interaction was detected between the rs6824447 polymorphism and the intake of oil in relation with insulin resistance, such that carriers of this minor allele who consumed sunflower oil had lower HOMA_IR than those who did not have this allele (P = 0.001). CONCLUSIONS: Genetic variations in the ELOVL6 gene were associated with insulin sensitivity in this population-based study.

Source inference of exogenous gamma-hydroxybutyric acid (GHB) administered to humans by means of carbon isotopic ratio analysis: novel perspectives regarding forensic investigation and intelligence issues.

γ-Hydroxybutyric acid (GHB) is an endogenous short-chain fatty acid popular as a recreational drug due to sedative and euphoric effects, but also often implicated in drug-facilitated sexual assaults owing to disinhibition and amnesic properties. Whilst discrimination between endogenous and exogenous GHB as required in intoxication cases may be achieved by the determination of the carbon isotope content, such information has not yet been exploited to answer source inference questions of forensic investigation and intelligence interests. However, potential isotopic fractionation effects occurring through the whole metabolism of GHB may be a major concern in this regard. Thus, urine specimens from six healthy male volunteers who ingested prescription GHB sodium salt, marketed as Xyrem(®), were analysed by means of gas chromatography/combustion/isotope ratio mass spectrometry to assess this particular topic. A very narrow range of δ(13)C values, spreading from -24.810/00 to -25.060/00, was observed, whilst mean δ(13)C value of Xyrem(®) corresponded to -24.990/00. Since urine samples and prescription drug could not be distinguished by means of statistical analysis, carbon isotopic effects and subsequent influence on δ(13)C values through GHB metabolism as a whole could be ruled out. Thus, a link between GHB as a raw matrix and found in a biological fluid may be established, bringing relevant information regarding source inference evaluation. Therefore, this study supports a diversified scope of exploitation for stable isotopes characterized in biological matrices from investigations on intoxication cases to drug intelligence programmes.

The lipid composition of rat brain aggregating cell cultures during development.

The lipid and fatty acid composition of rat brain was studied during its development both in vivo and in an aggregating cell culture system. Although the amount of lipid present in the cultures was very low, the increase in glycolipid content corresponded closely to the period of intense myelin formation. Very long chain fatty acids (hydroxylated and unsubstituted) were present in 41-day cultures. In comparison to the in vivo situation, myelination was delayed in vitro and, after 40 days in culture, cholesterol esters were 5-fold higher than in vivo, indicating that demyelination was occurring.

The Intestinal Microbiota Contributes to the Ability of Helminths to Modulate Allergic Inflammation.

Intestinal helminths are potent regulators of their host's immune system and can ameliorate inflammatory diseases such as allergic asthma. In the present study we have assessed whether this anti-inflammatory activity was purely intrinsic to helminths, or whether it also involved crosstalk with the local microbiota. We report that chronic infection with the murine helminth Heligmosomoides polygyrus bakeri (Hpb) altered the intestinal habitat, allowing increased short chain fatty acid (SCFA) production. Transfer of the Hpb-modified microbiota alone was sufficient to mediate protection against allergic asthma. The helminth-induced anti-inflammatory cytokine secretion and regulatory T cell suppressor activity that mediated the protection required the G protein-coupled receptor (GPR)-41. A similar alteration in the metabolic potential of intestinal bacterial communities was observed with diverse parasitic and host species, suggesting that this represents an evolutionary conserved mechanism of host-microbe-helminth interactions.

Análise de ácidos graxos por eletroforese capilar utilizando detecção condutométrica sem contato

This article describes a novel approach to the separation of fatty acids ranging from 8 to 20 carbons using capillary electrophoresis with contactless conductivity detection. Complete separation of nine linear chain fatty acids (from C8:0 to C20:0) was achieved in 15 min under normal polarity operation. Limits of detection ranged from 35 to 319 µmol L-1 for C20:0 to C8:0, respectively. The optimized running electrolyte composition was 5.0 mmol L-1 phosphate buffer at pH 7, 4.0 mmol L-1 dimethyl-b-cyclodextrin, 2.0 mmol L-1 trimethyl-b-cyclodextrin, acetonitrile 50% (v/v), and methanol 20% (v/v). The applicability of the separation system was demonstrated by the analysis of coconut vegetable oil.

Aplicação de lipases no tratamento de águas residuárias com elevados teores de lipídeos

The process of anaerobic digestion has been greatly developed during the last decades for the treatment of wastewater from food industries as for example, wastewaters from sugar and starch manufacturing and from breweries. However, for wastewaters which contain high amounts of fats and proteins, such as those from slaughterhouses and dairies, anaerobic digestion was found to be troublesome, due to the potential of sludge flotation, the formation of scum layers of lipids at the surface of the reactor, which are not digested and the toxicity of the intermediate compounds (long chain fatty acids) generated during the anaerobic digestion of the wastewater. The addition of hydrolytic enzymes, such as lipases prior to the anaerobic digestion can minimize these problems. In this work, this strategy is reviewed and the technical issues that must be considered in determining its feasibility are discussed.

Lipídios estruturados obtidos a partir da mistura de gordura de frango, sua estearina e triacilgliceróis de cadeia média: I- Composição em ácidos graxos e em triacilgliceróis

The purpose of this study is to analyze the interactions that occur in binary and ternary fat blends between medium and long chain triacylglycerols and their structured lipids obtained by chemical interesterification through the analysis of their physico-chemical properties. The synthesized structured triacylglycerols presented from 14.8 to 58.4% medium chain fatty acids, from 15.7 to 37.2% saturated fatty acids, from 19.2 to 47.5% monounsaturated fatty acids, and from 6.7 to 15.2% essential fatty acids. Chemical interesterification modified the behavior of binary and ternary mixtures and new types of triacylglycerol groups were formed.

Efficacy of the dietary histone deacetylase inhibitor butyrate alone or in combination with vitamin A against proliferation of MCF-7 human breast cancer cells

The combined treatment with histone deacetylase inhibitors (HDACi) and retinoids has been suggested as a potential epigenetic strategy for the control of cancer. In the present study, we investigated the effects of treatment with butyrate, a dietary HDACi, combined with vitamin A on MCF-7 human breast cancer cells. Cell proliferation was evaluated by the crystal violet staining method. MCF-7 cells were plated at 5 x 10(4) cells/mL and treated with butyrate (1 mM) alone or combined with vitamin A (10 µM) for 24 to 120 h. Cell proliferation inhibition was 34, 10 and 46% following treatment with butyrate, vitamin A and their combination, respectively, suggesting that vitamin A potentiated the inhibitory activities of butyrate. Furthermore, exposure to this short-chain fatty acid increased the level of histone H3K9 acetylation by 9.5-fold (Western blot), but not of H4K16, and increased the expression levels of p21WAF1 by 2.7-fold (Western blot) and of RARβ by 2.0-fold (quantitative real-time PCR). Our data show that RARβ may represent a molecular target for butyrate in breast cancer cells. Due to its effectiveness as a dietary HDACi, butyrate should be considered for use in combinatorial strategies with more active retinoids, especially in breast cancers in which RARβ is epigenetically altered.