Odd-chain Fatty Acids Research Articles

Analytical shortcomings and other considerations related to the identification of biomarkers of dairy fat intake

Over the last decade, the concentrations of certain odd-chain saturated fatty acids (for example, C15:0 and C17:0) and trans-palmitoleic acid (trans-9 C16:1) in plasma and erythrocytes have been proposed as biomarkers of dairy fat intake,1, 2, 3 which is expected to overcome major issues associated with dietary assessment methods that rely on self-report, such as food frequency questionnaires. However, some overlooked analytical issues may have resulted in misidentification or even precluded other candidate biomarkers of dairy fat intake from being identified in several previous studies.1, 2, 3, 4, 5 The separation of fatty acids in biological and foods matrices is usually performed by gas chromatography after lipid extraction and methylation/transesterification using either acid or alkali-catalysis.6 Fatty acid methyl esters (FAME) are routinely identified by retention time comparisons with authentic commercial standards using a gas chromatograph equipped with a flame ionization detector (GC-FID). Methyl esters (except geometric isomers) not available as commercial standards can be identified by GC-MS analysis of 4,4-dimethyloxazoline (DMOX) derivatives prepared from FAME as described elsewhere.7 However, achieving proper separation and unequivocal identification of FAME in foods containing a very complex lipid composition such as ruminant milk fat has proved particularly challenging due to partial or complete overlapping of a number of fatty acids even when the most appropriate columns (for example, CP-Sil 88, Varian Inc.; SP 2560, Supelco Inc.) and different GC temperature programs are used.8 The same shortcomings should be expected in the GC analysis of fatty acid composition from biological tissues collected from individuals consuming diets containing ruminant-derived foods such as dairy products, which could possibly lead to inaccurate analytical results and misleading conclusions.

The relationship between odd- and branched-chain fatty acids and microbial nucleic acid bases in rumen.

ObjectiveThis study aims to identify the relationship between odd- and branched-chain fatty acids (OBCFAs) and microbial nucleic acid bases in the rumen, and to establish a model to accurately predict microbial protein flow by using OBCFA.MethodsTo develop the regression equations, data on the rumen contents of individual cows were obtained from 2 feeding experiments. In the first experiment, 3 rumen-fistulated dry dairy cows arranged in a 3×3 Latin square were fed diets of differing forage to concentration ratios (F:C). The second experiment consisted of 9 lactating Holstein dairy cows of similar body weights at the same stage of pregnancy. For each lactation stage, 3 cows with similar milk production were selected. The rumen contents were sampled at 4 time points of every two hours after morning feeding 6 h, and then to analyse the concentrations of OBCFA and microbial nucleic acid bases in the rumen samples.ResultsThe ruminal bacteria nucleic acid bases were significantly influenced by feeding diets of differing forge to concentration ratios and lactation stages of dairy cows (p<0.05). The concentrations of OBCFAs, especially odd-chain fatty acids and C15:0 isomers, strongly correlated with the microbial nucleic acid bases in the rumen (p<0.05). The equations of ruminal microbial nucleic acid bases established by ruminal OBCFAs contents showed a good predictive capacity, as indicated by reasonably low standard errors and high R-squared values.ConclusionThis finding suggests that the rumen OBCFA composition could be used as an internal marker of rumen microbial matter.

Odd-chain fatty acids as a biomarker for dietary fiber intake: a novel pathway for endogenous production from propionate ,

Background: The risk of type 2 diabetes is inversely correlated with plasma concentrations of odd-chain fatty acids [OCFAs; pentadecanoic acid (15:0) and heptadecanoic acid (17:0)], which are considered as biomarkers for dairy fat intake in humans. However, rodent studies suggest that OCFAs are synthesized endogenously from gut-derived propionate. Propionate increases with dietary fiber consumption and has been shown to improve insulin sensitivity.Objective: We hypothesized that OCFAs are produced in humans from dietary fibers by a novel endogenous pathway.Design: In a randomized, double-blind crossover study, 16 healthy individuals were supplemented with cellulose (30 g/d), inulin (30 g/d), or propionate (6 g/d) for 7 d. In addition, human hepatoma cells were incubated with different propionate concentrations. OCFAs were determined in plasma phospholipids and hepatoma cells by gas chromatography.Results: Cellulose did not affect plasma OCFA levels, whereas inulin and propionate increased pentadecanoic acid by ∼17% (P < 0.05) and 13% (P = 0.05), respectively. The effect on heptadecanoic acid was even more pronounced, because it was elevated in almost all participants by inulin (11%; P < 0.01) and propionate (13%; P < 0.001). Furthermore, cell culture experiments showed a positive association between propionate and OCFA levels (R2 = 0.99, P < 0.0001), whereas palmitate (16:0) was negatively correlated (R2 = 0.83, P = 0.004).Conclusions: Our data show that gut-derived propionate is used for the hepatic synthesis of OCFAs in humans. The association of OCFAs with a decreased risk of type 2 diabetes may therefore also relate to dietary fiber intake and not only dairy fat. This trial was registered at www.germanctr.de as DRKS00010121.

Supplementation of glycerol or fructose via drinking water to grazing lambs on tissue glycogen level and lipogenesis.

Lambs ( = 18; 40.1 ± 7.4 kg BW) were used to assess supplementation of glycerol or fructose via drinking water on growth, tissue glycogen levels, postmortem glycolysis, and lipogenesis. Lambs were blocked by BW and allocated to alfalfa paddocks (2 lambs/paddock and 3 paddocks/treatment). Each paddock within a block was assigned randomly to drinking water treatments for 30 d: 1) control (CON), 2) 120 g fructose/L of drinking water (FRU), or 3) 120 g glycerol/L of drinking water (GLY). Lambs grazed alfalfa with free access to water treatments for 28 d and then were fasted in indoor pens for a final 2 d with access to only water treatments. Data were analyzed using the MIXED procedure of SAS with water treatment and time (when appropriate) in the model. During the 28-d grazing period, ADG was greater ( < 0.05) for GLY than for CON or FRU. During the 2-d fasting period, BW shrink was lower ( < 0.05) for GLY compared with CON or FRU. Hot carcass weight was greater ( < 0.05) for GLY than for FRU. The interaction for glycogen content × postmortem time was significant ( = 0.003) in LM and semitendinosus (ST) muscles. Glycogen content in the LM was greater ( < 0.05) for GLY at 2 and 3 h and for FRU at 1 h postmortem compared with CON. Glycogen content in ST did not differ between treatments ( > 0.05). Liver glycogen content was over 14-fold greater ( < 0.05) for GLY compared with FRU or CON. Liver free glucose was greater ( < 0.05) for GLY than for CON, whereas liver lipid content was higher ( < 0.05) for CON than for GLY. Supplementation with GLY increased ( < 0.05) odd-chain fatty acids in LM, subcutaneous fat (SQ), and the liver. Stearic acid (C18:0) concentrations were reduced in LM ( = 0.064) and subcutaneous adipose tissue (SQ; = 0.018), whereas oleic acid (C18:1 -9) concentration tended to be increased ( = 0.066) in SQ with FRU and GLY. Linolenic acid (C18:3 -3) was reduced ( = 0.031) and all long-chain -3 fatty acid (eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid) concentrations were increased ( < 0.05) with FRU and GLY compared with CON. Glycerol supplementation upregulated ( < 0.05) stearoyl-CoA desaturate () and fatty acid synthase () mRNA by over 40-fold in the SQ and 5-fold in the liver. Glycerol supplementation also upregulated ( < 0.05) glucose transporters and glycogen branching enzyme in the liver. Overall, glycerol supplementation improved growth, reduced BW shrink during fasting, increased glycogen content in muscle and the liver, and stimulated de novo lipogenesis.

Even- and odd-chain saturated fatty acids in serum phospholipids are differentially associated with adipokines.

BackgroundSaturated fatty acids are generally thought to have detrimental effects on health. However, a recent study showed that even- and odd-chain saturated fatty acids had opposite associations with type 2 diabetes. Limited studies of Western populations examined the associations of circulating saturated fatty acids with adipokines, an important role in glucose metabolism.ObjectiveWe examined the associations of saturated fatty acids in serum phospholipids with circulating levels of adipokines among a Japanese population.DesignA cross-sectional study was conducted among 484 Japanese employees (284 men and 200 women) aged 20–65 years. The serum fatty acid composition in the phospholipid fraction was measured by gas-chromatography. Serum leptin, adiponectin, plasminogen activator inhibitor-1 (PAI-1), resistin, and visfatin were measured using a Luminex suspension bead-based multiplexed array. Multiple linear regression analysis was performed to assess the association between saturated fatty acids and adipokines, with adjustment for potential confounding variables.ResultsEven- and odd-chain saturated fatty acids were differentially associated with adipokines. Higher levels of even-chain saturated fatty acids (14:0 myristic, 16:0 palmitic, and 18:0 stearic acids) were associated with higher levels of resistin (P for trend = 0.048) and lower levels of adiponectin (P for trend = 0.003). By contrast, odd-chain saturated fatty acids (15:0 pentadecanoic and 17:0 heptadecanoic acids) showed inverse associations with leptin and PAI-1 (P for trend = 0.048 and 0.02, respectively). Visfatin was positively associated with both even- and odd-chain saturated fatty acids.ConclusionsThe results suggest that even- and odd-chain saturated fatty acids are differentially associated with adipokine profile.

Odd Chain Fatty Acids; New Insights of the Relationship Between the Gut Microbiota, Dietary Intake, Biosynthesis and Glucose Intolerance

Recent findings have shown an inverse association between circulating C15:0/C17:0 fatty acids with disease risk, therefore, their origin needs to be determined to understanding their role in these pathologies. Through combinations of both animal and human intervention studies, we comprehensively investigated all possible contributions of these fatty acids from the gut-microbiota, the diet, and novel endogenous biosynthesis. Investigations included an intestinal germ-free study and a C15:0/C17:0 diet dose response study. Endogenous production was assessed through: a stearic acid infusion, phytol supplementation, and a Hacl1−/− mouse model. Two human dietary intervention studies were used to translate the results. Finally, a study comparing baseline C15:0/C17:0 with the prognosis of glucose intolerance. We found that circulating C15:0/C17:0 levels were not influenced by the gut-microbiota. The dose response study showed C15:0 had a linear response, however C17:0 was not directly correlated. The phytol supplementation only decreased C17:0. Stearic acid infusion only increased C17:0. Hacl1−/− only decreased C17:0. The glucose intolerance study showed only C17:0 correlated with prognosis. To summarise, circulating C15:0 and C17:0 are independently derived; C15:0 correlates directly with dietary intake, while C17:0 is substantially biosynthesized, therefore, they are not homologous in the aetiology of metabolic disease. Our findings emphasize the importance of the biosynthesis of C17:0 and recognizing its link with metabolic disease.

Novel Odd-Chain Fatty Acids with a Terminal Double Bond in Ovaries of the Limpet Cellana toreuma.

Our previous study characterized highly diverse dienoic fatty acids (FA), in particular an uncommon non-methylene-interrupted (NMI) FA, in the ovaries of the Japanese limpet Cellana toreuma belonging to the archaeogastropods, but many minor chemically unidentified FA remain. In this study, among previously unidentified FA (less than 0.1% of total FA), four novel NMI FA with a terminal double bond [7,18-nonadecadienoic (19:2Δ7,18), 11,18-nonadecadienoic (19:2Δ11,18), 7,20-heneicosadienoic (21:2Δ7,20), and 11,20-heneicosadienoic (21:2Δ11,20) acids] were found, along with known 14-pentadecenoic (15:1Δ14), 16-heptadecenoic (17:1Δ16), and 9,18-nonadecadienoic (19:2Δ9,18) acids, based on capillary GC-MS of their methyl esters, 3-pyridylcarbinol derivatives, and argentation thin-layer chromatography. From our findings, possible biosynthetic pathways for the novel FA are discussed.

The Lipid Composition of Subcutaneous Adipose Tissue of Brown Bears (Ursus arctos) in Croatia.

The composition of adipose tissue in brown bears (Ursus arctos) is highly variable and depends on an individual's feeding habits. Fatty acid composition of subcutaneous adipose tissue (SAT) may provide insight into brown bear feeding habits, for which data are scarce. The aim of this study was to determine the lipid composition of SAT and variations in the composition of fatty substances with regard to gender and to assess SAT relative to season and body mass (BM) of brown bears in Croatia. Seventy-six tissue samples of brown bear SAT were analyzed in this study. We found that gender, season, and BM significantly affected the lipid composition of SAT. Both females and males had higher percentages of saturated fatty acids and monounsaturated fatty acids (MUFAs) in SAT in spring than in autumn, while the percentage of polyunsaturated fatty acids (PUFAs) was higher in autumn. The prevalence of MUFAs in SAT and the greater presence of PUFAs in autumn, together with the presence of odd-chain saturated fatty acids, indicate the importance of these fatty acids in brown bear physiology. We suggest that the lipid content of adipose tissue may provide valuable information on changes in brown bear condition in response to feeding habits and the effects of supplemental feeding.

Environmental Enteric Dysfunction is Associated with Carnitine Deficiency and Altered Fatty Acid Oxidation

BackgroundEnvironmental enteric dysfunction (EED), a condition characterized by small intestine inflammation and abnormal gut permeability, is widespread in children in developing countries and a major cause of growth failure. The pathophysiology of EED remains poorly understood. MethodsWe measured serum metabolites using liquid chromatography-tandem mass spectrometry in 400 children, aged 12–59months, from rural Malawi. Gut permeability was assessed by the dual-sugar absorption test. Findings80.7% of children had EED. Of 677 serum metabolites measured, 21 were negatively associated and 56 were positively associated with gut permeability, using a false discovery rate approach (q<0.05, p<0.0095). Increased gut permeability was associated with elevated acylcarnitines, deoxycarnitine, fatty acid β-oxidation intermediates, fatty acid ω-oxidation products, odd-chain fatty acids, trimethylamine-N-oxide, cystathionine, and homocitrulline, and with lower citrulline, ornithine, polyphenol metabolites, hippurate, tryptophan, and indolelactate. InterpretationEED is a syndrome characterized by secondary carnitine deficiency, abnormal fatty acid oxidation, alterations in polyphenol and amino acid metabolites, and metabolic dysregulation of sulfur amino acids, tryptophan, and the urea cycle. Future studies are needed to corroborate the presence of secondary carnitine deficiency among children with EED and to understand how these metabolic derangements may negatively affect the growth and development of young children.

Milk fat biomarkers and cardiometabolic disease.

Purpose of reviewDairy is a major food group with potential impact on cardiometabolic health. Self-reported dairy intake has limitations that can partly be avoided by using biomarkers. This review aims to summarize the evidence of odd-chain saturated fatty acids (OCFAs), that is, pentadecanoic acid (C15 : 0) and heptadecanoic acid (17 : 0), as biomarkers of dairy fat intake. In addition, the associations of OCFA biomarkers with cardiometabolic disease will be overviewed.Recent findingsAdipose tissue 15 : 0 is the preferred biomarker but also circulating 15 : 0, and to a weaker extent 17 : 0, reflects both habitual and changes in dairy intake. Whereas results from studies assessing cardiovascular outcomes are inconsistent, OCFA biomarkers are overall associated with lower diabetes risk. Residual confounding should however be considered until interventional data and mechanisms are available. Although OCFA biomarkers mainly reflect dairy fat intake, recently proposed endogenous synthesis and metabolism do motivate further research.SummaryTaking into account the study population diet and limitations of OCFA biomarkers, both adipose and circulating levels of 15 : 0, in particular, are useful for estimating total dairy fat intake. OCFA biomarkers are overall not linked to cardiovascular disease risk, but a possible beneficial role of dairy foods in diabetes prevention warrant further study.

Critical role of the peroxisomal protein PEX16 in white adipocyte development and lipid homeostasis

The importance of peroxisomes for adipocyte function is poorly understood. Herein, we provide insights into the critical role of peroxin 16 (PEX16)-mediated peroxisome biogenesis in adipocyte development and lipid metabolism. Pex16 is highly expressed in adipose tissues and upregulated during adipogenesis of murine and human cells. We demonstrate that Pex16 is a target gene of the adipogenesis “master-regulator” PPARγ. Stable silencing of Pex16 in 3T3-L1 cells strongly reduced the number of peroxisomes while mitochondrial number was unaffected. Concomitantly, peroxisomal fatty acid (FA) oxidation was reduced, thereby causing accumulation of long- and very long-chain (polyunsaturated) FAs and reduction of odd-chain FAs. Further, Pex16-silencing decreased cellular oxygen consumption and increased FA release. Additionally, silencing of Pex16 impaired adipocyte differentiation, lipogenic and adipogenic marker gene expression, and cellular triglyceride stores. Addition of PPARγ agonist rosiglitazone and peroxisome-related lipid species to Pex16-silenced 3T3-L1 cells rescued adipogenesis. These data provide evidence that PEX16 is required for peroxisome biogenesis and highlights the relevance of peroxisomes for adipogenesis and adipocyte lipid metabolism.

Milk fatty acid profile in cows fed red clover- or alfalfa-silage based diets differing in rumen-degradable protein supply

Polyphenol oxidase in red clover (RC) silage reduces lipolysis and consequently protects its constituent fatty acids (FA) against biohydrogenation by ruminal microorganisms. Fatty acid biohydrogenation could be further inhibited by reducing the nitrogen (N) supply to ruminal bacteria. To compare the effects of RC and alfalfa (AL) silage fed in diets differing in rumen-degradable protein supply on the transfer efficiency of polyunsaturated FA from diet to milk, and on the resulting FA profile of milk fat, 8 multiparous Holstein dairy cows (72±17days in milk) were used in a replicated 4×4 Latin square design (21-day periods including 14days of adaptation). Four treatments were compared in a 2×2 factorial arrangement with AL or RC fed in diets formulated to provide 100% (RDP‐100) or 85% (RDP‐85) of calculated rumen-degradable protein requirements. Rumen-degradable protein concentrations were adjusted by varying the supplies of ground vs. steam-flaked corn grains, and untreated vs. heat-treated soybean meals. Feeding RC, as compared with AL diets, decreased ruminal ammonia-N concentration as well as urea-N in plasma and milk. Intakes of c9c12 18:2 and c9c12c15 18:3 were lower for cows fed RC, but their secretion in milk and their apparent transfer efficiency from diet were greater as compared to cows fed AL. As a result, feeding RC increased the concentrations of c9c12 18:2 and c9c12c15 18:3, but decreased the proportions of t11 18:1, c9t11 18:2, and t11c15 18:2 in milk fat. These results are consistent with the lower effective ruminal disappearance of c9c12 18:2 and c9c12c15 18:3 in RC as determined by a 96h in sacco incubation, and the lower abundance of ruminal bacteria capable of hydrolyzing dietary lipids and hydrogenating polyunsaturated FA (Ruminococcus albus and Ruminococcus flavefaciens) in the rumen content. The apparent transfer efficiency of c9c12 18:2 from diet to milk was further increased by feeding RDP‐85 as compared with RDP‐100 diet. Feeding RC decreased concentrations of linear odd-chain FA (11:0, 13:0, 15:0, 17:0, and c9 17:1) and increased proportions of branched-chain FA (iso13:0, iso14:0, iso15:0, iso16:0, iso17:0, anteiso15:0, and anteiso17:0) in milk fat as compared with AL. In conclusion, as compared with AL, cows fed RC produced milk with greater concentrations of major forage FA (c9c12 18:2 and c9c12c15 18:3) and lower proportions of intermediates in ruminal biohydrogenation of these FA. Variations in milk fat concentrations of odd- and branched-chain FA, which are synthesized in the rumen by various microbial populations, may be reflecting the effects of forage source on ruminal fermentation.

418 - Odd-Chain Fatty Acids Predict Insulin Sensitivity in People with T2DM and Protect HepG2 Cells from Palmitate-Induced Insulin Resistance Via PPARα

Lipid-induced dysfunction of the liver, especially steatohepatitis, is becoming increasingly prevalent and may be an important link between obesity and type 2 diabetes (T2DM). Elevated circulating saturated fatty acids contribute to steatohepatitis and T2DM while other plasma free fatty acids appear protective. Activated PPARα is involved in regulating hepatic lipid accumulation by upregulating genes responsible for fatty acid transport and metabolism. We examined the hypothesis that specific fatty acids predict insulin sensitivity in T2DM patients and modulate risk of steatohepatitis by activating PPARα. The plasma fatty acid profile of patients with T2DM (n=43) and healthy control subjects (n=45) was analysed using gas chromatography. An in vitro model of terminally differentiated (d)HepG2 cells was used to determine the mechanism of effect of novel protective fatty acids on insulin resistance and hepatocyte dysfunction. Plasma levels of odd-chain saturated fatty acids (OCSFA) predicted fasting plasma glucose in a multiple regression model, after adjusting for age, BMI and gender. OCSFA protected dHepG2 cells from palmitate-induced insulin resistance and cellular dysfunction; by increasing cellular glycogen content, cellular monounsaturated fatty acid (MUFA) concentration and reducing output of glucose and TNFα. The PPARα antagonist GW6471 blocked the increase of MUFA due to OCSFA in palmitate-exposed hepatocytes. Furthermore, OCSFA increased PPARα activity in palmitate-exposed hepatocytes more than the PPARα agonist fenofibrate. OCSFA induced upregulation of the PPARα targets, stearoyl Co-A desaturase 1 and fatty acid synthase in palmitate-exposed hepatocytes. In summary, OCSFA inversely associates with insulin resistance and promotes MUFA accumulation via a PPARα-dependent pathway, exerting a protective effect against hepatocyte dysfunction.

The effect of cattle breed, season and type of diet on the fatty acid profile of raw milk

Abstract. The aim of the study was to determine the effect of cow breed, season and type of diet on the fatty acid (FA) profile of raw milk. A 2-year study was conducted on bulk milk samples collected from eight herds consisting of Czech Fleckvieh (CF, four herds) and Holstein (H, four herds) breeds. One half of the herds of each breed was grazed (G), while the other half was not (N). Samples were collected twice in winter (W) and twice in summer (S). Milk yield in CF (5385.50 kg) was lower than in H (7015.15 kg, P &lt; 0.05). The effect of breed was found in odd-chain, branch-chain and hypercholesterolemic FAs (P &lt; 0.05). The content of fat was lower in summer (S) than in winter (W), being 3.71 and 3.91 g 100 g−1, respectively (P &lt; 0.05). The proportion of saturated and polyunsaturated FAs was lower in S than in W (P &lt; 0.05). The content of monounsaturated FAs was higher in S (30.69 g 100 g−1) than in W (27.72 g 100 g−1, P &lt; 0.05). Milk yield in grazing herds (G, 5197.50 kg) was lower (P &lt; 0.05) than in non-grazing herds (N, 7203.75 kg). The sum of saturated and hypercholesterolemic FAs was lower and the sum of monounsaturated and odd-chain FAs was higher in G than in N (P &lt; 0.05). Content of conjugated linoleic acid (CLA) and C18:3n3 was higher in G (0.93 and 0.64 g 100 g−1) than in N (0.42 and 0.39 g 100 g−1, respectively, P &lt; 0.001).

Novel Mouse Models of Methylmalonic Aciduria Recapitulate Phenotypic Traits with a Genetic Dosage Effect

Methylmalonic aciduria (MMAuria), caused by deficiency of methylmalonyl-CoA mutase (MUT), usually presents in the newborn period with failure to thrive and metabolic crisis leading to coma or even death. Survivors remain at risk of metabolic decompensations and severe long term complications, notably renal failure and neurological impairment. We generated clinically relevant mouse models of MMAuria using a constitutive Mut knock-in (KI) allele based on the p.Met700Lys patient mutation, used homozygously (KI/KI) or combined with a knockout allele (KO/KI), to study biochemical and clinical MMAuria disease aspects. Transgenic Mut(ki/ki) and Mut(ko/ki) mice survive post-weaning, show failure to thrive, and show increased methylmalonic acid, propionylcarnitine, odd chain fatty acids, and sphingoid bases, a new potential biomarker of MMAuria. Consistent with genetic dosage, Mut(ko/ki) mice have lower Mut activity, are smaller, and show higher metabolite levels than Mut(ki/ki) mice. Further, Mut(ko/ki) mice exhibit manifestations of kidney and brain damage, including increased plasma urea, impaired diuresis, elevated biomarkers, and changes in brain weight. On a high protein diet, mutant mice display disease exacerbation, including elevated blood ammonia, and catastrophic weight loss, which, in Mut(ki/ki) mice, is rescued by hydroxocobalamin treatment. This study expands knowledge of MMAuria, introduces the discovery of new biomarkers, and constitutes the first in vivo proof of principle of cobalamin treatment in mut-type MMAuria.

A multi-analytical approach applied to the archaeological residues in Iberian glasses. Earliest evidences on the consumption of fermented beverages in votive rituals

One of the analytical difficulties that may appear in the analytical process of the archaeological materials is related to the complexity of matrices. In this sense, the abundant content of lipids in archaeological degraded material makes the analysis of polyphenols much more problematic. In addition, phenolic compounds with a low-molecular weight might be present at trace level or even not be present in ancient artefacts. In this study, the advantages of gas chromatography–mass spectrometry in selected ion monitoring mode (GC–MS-SIM) for the analysis of wine biomarkers, tartaric and cinnamic acids, preserved in archaeological potsherd found in a votive deposit from the Iberian period in Granada (Spain) are presented. This will contribute to a greater understanding of the consumption of fermented beverages offered in votive rituals in Southern Spain during the Iberian period. To our knowledge, this is the first time that GC–MS-SIM technique is used to identify low concentrations of these polyphenolic acids in archaeological residues. In addition, the GC–MS-SCAN and gas chromatography–combustion-isotope ratio mass spectrometry (GC–C–IRMS) analysis revealed the presence of animal fats and vegetable oils mixed with wax and conifer resin preserved in the Iberian glasses. The correlation between organic compounds identified in the residues (short and odd-chain fatty acids (C9:0 to C12:0, C17:0 and C19:0), palmitic, stearic, azelaic, oleic, lignoceric and dehydroabietic acids and long-chain odd-numbered alkanes) and its possible origin are also discussed.

A comprehensive study of serum odd‐ and branched‐chain fatty acids in patients with excess weight

While small amounts of odd-chain fatty acids (OCFAs) and branched-chain fatty acids (BCFAs) were known to be present in mammals, it was quite recently that they were shown to play an important role in human health. However, still little is known on OCFA and BCFA profiles in subjects who have obesity. The aim of this study was to verify whether obesity is associated with changes in serum OCFA and BCFA profiles. Serum content of fatty acids was determined by gas chromatography-mass spectroscopy in 23 patients with excess weight and 21 nonobese controls. Six OCFAs and six BCFAs (three iso-BCFAs and three anteiso-BCFAs) were found in sera from the examined subjects. Patients with excess weight presented with significantly lower serum iso-BCFA levels than the controls. Total serum content of iso-BCFAs correlated inversely with serum insulin, triglycerides, and 18:1/18:0 desaturation index. Both OCFA and iso-BCFA levels correlated inversely with C-reactive protein concentration. Lower iso-BCFA content in patients with excess weight may be involved in elevation of serum concentration of triglycerides and inflammation. Decreased contents of iso-BCFAs in subjects with have obesity, and established anti-inflammatory, antidiabetic, and anticancer properties of these fatty acids, point to potential beneficial effects of an iso-BCFA-rich diet.

Differential RNA-seq, Multi-Network Analysis and Metabolic Regulation Analysis of Kluyveromyces marxianus Reveals a Compartmentalised Response to Xylose.

We investigated the transcriptomic response of a new strain of the yeast Kluyveromyces marxianus, in glucose and xylose media using RNA-seq. The data were explored in a number of innovative ways using a variety of networks types, pathway maps, enrichment statistics, reporter metabolites and a flux simulation model, revealing different aspects of the genome-scale response in an integrative systems biology manner. The importance of the subcellular localisation in the transcriptomic response is emphasised here, revealing new insights. As was previously reported by others using a rich medium, we show that peroxisomal fatty acid catabolism was dramatically up-regulated in a defined xylose mineral medium without fatty acids, along with mechanisms to activate fatty acids and transfer products of β-oxidation to the mitochondria. Notably, we observed a strong up-regulation of the 2-methylcitrate pathway, supporting capacity for odd-chain fatty acid catabolism. Next we asked which pathways would respond to the additional requirement for NADPH for xylose utilisation, and rationalised the unexpected results using simulations with Flux Balance Analysis. On a fundamental level, we investigated the contribution of the hierarchical and metabolic regulation levels to the regulation of metabolic fluxes. Metabolic regulation analysis suggested that genetic level regulation plays a major role in regulating metabolic fluxes in adaptation to xylose, even for the high capacity reactions, which is unexpected. In addition, isozyme switching may play an important role in re-routing of metabolic fluxes in subcellular compartments in K. marxianus.

Effect of lipid supplementation on milk odd- and branched-chain fatty acids in dairy cows

Eight ruminally fistulated, multiparous Holstein cows were arranged in a double 4×4 Latin square with 14-d periods to investigate the effects of lipid supplementation on performance, rumen parameters, the milk odd- and branched-chain fatty acid (OBCFA) profile, and the relationships between milk OBCFA and rumen parameters. Lipid supplementation is known to inhibit microbial growth in the rumen, decrease de novo microbial fatty acid synthesis, and increase the uptake of circulating fatty acids by the mammary gland; treatments were selected to isolate these effects on the milk OBCFA profile. The 4 treatments were (1) a lipid-free emulsion medium infused in the rumen (CTL), (2) soybean oil as a source of polyunsaturated fatty acids infused in the rumen (RSO), (3) saturated fatty acids (38% 16:0, 40% 18:0) infused in the rumen (RSF), and (4) saturated fatty acids infused in the abomasum (ASF). Fat supplements were provided continuously as emulsions at a rate of 450g/d. Preplanned contrasts compared CTL to RSO, RSO to RSF, and RSF to ASF. Infusing RSO slightly decreased ruminal pH, but did not affect volatile fatty acids profile and milk fat concentration as compared with CTL. The yields of energy-corrected milk, fat, and protein were greater with RSF compared with RSO. The concentration of odd-chain fatty acids was decreased by RSO, whereas even-chain iso fatty acids were not affected. Milk fat concentration of 17:0 + cis-9 17:1 was higher for RSF than for RSO, due to the saturated fatty acids supplement containing 2% 17:0 + cis-9 17:1. Limited differences were observed in the milk OBCFA profile between RSF and ASF. A multiple regression analysis yielded the following equation for predicting rumen pH based on milk fatty acids: pH=6.24 – (0.56×4:0) + (1.67 × iso 14:0) + (4.22 × iso 15:0) + (9.41×22:0). Rumen propionate concentration was negatively correlated with milk fat concentration of iso 14:0 and positively correlated with milk 15:0, whereas the acetate-to-propionate ratio gave the opposite correlations with milk iso 14:0 and 15:0. Milk fat concentration of 17:0 + cis-9 17:1 was not related to rumen propionate or to acetate-to-propionate ratio, due to the presence of 17:0 and cis-9 17:1 in the saturated fatty acids supplement. The results suggest that although lipid supplementation can affect the profile of milk OBCFA, the promise remains of using these milk fatty acids to evaluate rumen function.

359. Targeted Genome Editing Using TALENs to Correct a Mouse Model of Methylmalonic Acidemia (MMA)

Methylmalonic acidemia (MMA) is an autosomal recessive metabolic disorder, in which the body is unable to oxidize valine and isoleucine as well as odd chain fatty acids. Approximately 60 percent of MMA cases are caused by mutations in methylmalonyl-CoA mutase (MUT), the enzyme that catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA. Current treatments for MUT class MMA include dietary protein restriction and cofactor supplementation; however patients continue to exhibit severe morbidity and high rates of mortality. A murine model of MMA was created by knocking out exon 3 of the Mut gene and exhibits the severest clinical phenotype of MMA, neonatal lethality. Gene therapy treatments using recombinant adeno-associated viral vectors to treat mice with MMA have proven effective, however long-term follow-up of mice treated as neonates revealed genotoxicity caused by AAV integrations, leading the mice to develop hepatocellular carcinoma. Programmable nucleases such as TALENs should make it possible to correct at the MUT locus, ameliorate disease symptoms and reduce the effects of off-target integration by precise genome modification. Furthermore, AAV-mediated delivery of TALENs with an HR cassette carrying a rescue cDNA for the Mut gene in a disease model of MMA should enable in vivo correction in the liver, and ameliorate the MMA phenotype. We have designed TALENs that target intron 2 of the Mut gene and a partial rescue cassette that contains codon-optimized exons 3 to 13 of the Mut gene to restore Mut expression in the exon 3 knock out model of MMA after successful homologous recombination. The TALENs are expressed under the control of the liver-specific thyroxine binding globulin promoter to ensure cleavage in hepatocytes, and delivered using a hepatotropic AAV serotype 8 capsid. To restore expression and achieve targeted integration, the rescue cassette is preceded by a splice acceptor site and flanked by homology arms around the TALEN cleavage site. Integration of the rescue cassette will restore Mut expression at the locus, using the endogenous promoter and exons 1 and 2 of the Mut gene to reconstitute a functional Mut gene. AAV packaging limitations necessitate two AAV vectors for delivery of the TALENs, which are 4kb per pair, and required regulatory elements, as well as a third vector for delivery of the Mut homologous recombination rescue cassette. All three vectors will be delivered systemically in the neonatal period. We hypothesize that in vivo editing at the Mut locus with subsequent HR will provide sufficient Mut enzyme activity to improve the clinical and biochemical phenotypes of the MMA mice, with minimal off-target integration, thereby decreasing the risk of genotoxicity associated with AAV gene therapy.