Effects of dietary carbohydrate on hepatic de novo lipogenesis in European seabass (Dicentrarchus labrax L.)
2016; Elsevier BV; Volume: 57; Issue: 7 Linguagem: Inglês
10.1194/jlr.m067850
ISSN1539-7262
AutoresIván Viegas, Ivana Jarak, João Rito, Rui A. Carvalho, Isidoro Metón, Miguel Ã. Pardal, Isabel V. Baanante, John G. Jones,
Tópico(s)Reproductive biology and impacts on aquatic species
ResumoFarmed seabass have higher adiposity than their wild counterparts and this is often attributed to carbohydrate (CHO) feeding. Whether this reflects a reduction in fat oxidation, increased de novo lipogenesis (DNL), or both, is not known. To study the effects of high CHO diets on hepatic TG biosynthesis, hepatic TG deuterium (2H) enrichment was determined following 6 days in 2H-enriched tank water for fish fed with a no-CHO control diet (CTRL), and diets with digestible starch (DS) and raw starch (RS). Hepatic fractional synthetic rates (FSRs, percent per day−1) were calculated for hepatic TG-glyceryl and FA moieties through 2H NMR analysis. Glyceryl FSRs exceeded FA FSRs in all cases, indicating active cycling. DS fish did not show increased lipogenic potential compared to CTRL. RS fish had lower glyceryl FSRs compared with the other diets and negligible levels of FA FSRs despite similar hepatic TG levels to CTRL. DS-fed fish showed higher activity for enzymes that can provide NADPH for lipogenesis, relative to CTRL in the case of glucose-6-phosphate dehydrogenase (G6PDH) and relative to RS for both G6PDH and 6-phosphogluconate dehydrogenase. This approach indicated that elevated hepatic adiposity from DS feeding was not attributable to increased DNL. Farmed seabass have higher adiposity than their wild counterparts and this is often attributed to carbohydrate (CHO) feeding. Whether this reflects a reduction in fat oxidation, increased de novo lipogenesis (DNL), or both, is not known. To study the effects of high CHO diets on hepatic TG biosynthesis, hepatic TG deuterium (2H) enrichment was determined following 6 days in 2H-enriched tank water for fish fed with a no-CHO control diet (CTRL), and diets with digestible starch (DS) and raw starch (RS). Hepatic fractional synthetic rates (FSRs, percent per day−1) were calculated for hepatic TG-glyceryl and FA moieties through 2H NMR analysis. Glyceryl FSRs exceeded FA FSRs in all cases, indicating active cycling. DS fish did not show increased lipogenic potential compared to CTRL. RS fish had lower glyceryl FSRs compared with the other diets and negligible levels of FA FSRs despite similar hepatic TG levels to CTRL. DS-fed fish showed higher activity for enzymes that can provide NADPH for lipogenesis, relative to CTRL in the case of glucose-6-phosphate dehydrogenase (G6PDH) and relative to RS for both G6PDH and 6-phosphogluconate dehydrogenase. This approach indicated that elevated hepatic adiposity from DS feeding was not attributable to increased DNL. In aquaculture, there is high interest in substituting fishmeal protein with carbohydrate (CHO)-based substrates such as vegetable starch. Procurement of fishmeal protein remains highly dependent on overexploited wild fisheries (1Kristofersson D. Anderson J.L. Is there a relationship between fisheries and farming? Interdependence of fisheries, animal production and aquaculture.Mar. Policy. 2006; 30: 721-725Crossref Scopus (86) Google Scholar); hence any reduction in its consumption by farmed fish would reduce the ecological burden and improve the sustainability of aquaculture (2Gatlin D.M. Barrows F.T. Brown P. Dabrowski K. Gaylord T.G. Hardy R.W. Herman E. Hu G. Krogdahl Å. Nelson R. et al.Expanding the utilization of sustainable plant products in aquafeeds: a review.Aquacult. Res. 2007; 38: 551-579Crossref Scopus (1528) Google Scholar). Furthermore, to the extent that dietary CHO replaces protein for systemic glucose and energy demands (3Viegas I. Rito J. Jarak I. Leston S. Caballero-Solares A. Metón I. Pardal M.A. Baanante I.V. Jones J.G. Contribution of dietary starch to hepatic and systemic carbohydrate fluxes in European seabass (Dicentrarchus labrax L.).Br. J. Nutr. 2015; 113: 1345-1354Crossref PubMed Scopus (18) Google Scholar), it decreases waste ammonia generation from protein catabolism, thereby reducing nitrogenous effluents. For carnivorous fish such as the European seabass (Dicentrarchus labrax L.), the efficacy of this approach depends on the capacity of the fish to adapt from their natural diet that is high in both protein and fat, but lacking in CHO, to a regime where the proportion of dietary CHO to total caloric content is increased (4Enes P. Panserat S. Kaushik S. Oliva-Teles A. Dietary carbohydrate utilization by European sea bass (Dicentrarchus labrax L.) and gilthead sea bream (Sparus aurata L.) juveniles.Rev. Fish. Sci. 2011; 19: 201-215Crossref Scopus (104) Google Scholar). The capacity to digest complex CHO varies widely between different fish species (5Krogdahl Å. Hemre G.I. Mommsen T.P. Carbohydrates in fish nutrition: digestion and absorption in postlarval stages.Aquacult. Nutr. 2005; 11: 103-122Crossref Scopus (436) Google Scholar). Generally, carnivorous fish are poorly able to digest raw starch (RS). Cooking or gelatinizing the starch significantly improves its digestibility (6Dias J. Alvarez M.J. Diez A. Arzel J. Corraze G. Bautista J.M. Kaushik S.J. Regulation of hepatic lipogenesis by dietary protein/energy in juvenile European seabass (Dicentrarchus labrax).Aquaculture. 1998; 161: 169-186Crossref Scopus (254) Google Scholar, 7Peres H. Oliva-Teles A. Utilization of raw and gelatinized starch by European sea bass (Dicentrarchus labrax) juveniles.Aquaculture. 2002; 205: 287-299Crossref Scopus (168) Google Scholar), in part through stimulating α-amylase expression and activity (8Péres A. Zambonino Infante J.L. Cahu C. Dietary regulation of activities and mRNA levels of trypsin and amylase in sea bass (Dicentrarchus labrax) larvae.Fish Physiol. Biochem. 1998; 19: 145-152Crossref Scopus (169) Google Scholar). 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Temperature and dietary carbohydrate level effects on performance and metabolic utilisation of diets in European sea bass (Dicentrarchus labrax) juveniles.Aquaculture. 2008; 274: 153-160Crossref Scopus (190) Google Scholar), which is generally detrimental to flesh quality and market value (13Izquierdo M.S. Obach A. Arantzamendi L. Montero D. Robaina L. Rosenlund G. Dietary lipid sources for seabream and seabass: growth performance, tissue composition and flesh quality.Aquacult. Nutr. 2003; 9: 397-407Crossref Scopus (316) Google Scholar). CHO intake in seabass also increases lipid retention leading to lipid accumulation in the liver and viscera (14Castro C. Corraze G. Pérez-Jiménez A. Larroquet L. Cluzeaud M. Panserat S. Oliva-Teles A. Dietary carbohydrate and lipid source affect cholesterol metabolism of European sea bass (Dicentrarchus labrax) juveniles.Br. J. Nutr. 2015; 114: 1143-1156Crossref PubMed Scopus (45) Google Scholar). Given that high CHO intake promotes hepatic de novo lipogenesis (DNL) in mammals, it was hypothesized that, for farmed seabass, a diet with a high digestible starch (DS) content would also promote hepatic DNL activity and might, in part, explain the increased adiposity associated with dietary CHO supplementation. To test this hypothesis, we estimated DNL by measuring incorporation of deuterated water (2H2O) into hepatic TG using 2H NMR as previously described for mammals (15Delgado T.C. Pinheiro D. Caldeira M. Castro M.M.C.A. Geraldes C.F.G.C. López-Larrubia P. Cerdán S. Jones J.G. Sources of hepatic triglyceride accumulation during high-fat feeding in the healthy rat.NMR Biomed. 2009; 22: 310-317Crossref PubMed Scopus (46) Google Scholar, 16Duarte J.A.G. Carvalho F. Pearson M. Horton J.D. Browning J.D. Jones J.G. Burgess S.C. A high-fat diet suppresses de novo lipogenesis and desaturation but not elongation and triglyceride synthesis in mice.J. Lipid Res. 2014; 55: 2541-2553Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 17Soares A.F. Carvalho R.A. Veiga F.J. Alves M.G. Martins F.O. Viegas I. González J.D. Metón I. Baanante I.V. Jones J.G. Restoration of direct pathway glycogen synthesis flux in the STZ-diabetes rat model by insulin administration.Am. J. Physiol. Endocrinol. Metab. 2012; 303: E875-E885Crossref PubMed Scopus (18) Google Scholar). For fish metabolic studies, 2H2O can be conveniently incorporated into tank water without disturbing feeding and behavior (18Gasier H.G. Previs S.F. Pohlenz C. Fluckey J.D. Gatlin D.M. Buentello J.A. A novel approach for assessing protein synthesis in channel catfish, Ictalurus punctatus.Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2009; 154: 235-238Crossref PubMed Scopus (22) Google Scholar, 19Viegas I. Mendes V.M. Leston S. Jarak I. Carvalho R.A. Pardal M.A. Manadas B. Jones J.G. Analysis of glucose metabolism in farmed European sea bass (Dicentrarchus labrax L.) using deuterated water.Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2011; 160: 341-347Crossref PubMed Scopus (27) Google Scholar). We applied this method to a group of fish fed on a control CHO-free diet and a second group fed a diet supplemented with gelatinized starch. To determine whether starch digestibility had an independent effect on DNL activity, a third group of fish fed a diet supplemented with RS was also studied. European seabass (D. labrax L.) provided by Tinamenor (Cantabria, Spain) were transported to the laboratory and randomly assigned to three different tanks (n = 20–32 per tank; initial mean length of 10.8 ± 0.5 cm and initial mean body weight of 21.9 ± 0.3 g). Fish were acclimated at 20°C and 30‰ salinity in 200 l tanks supplied with well-aerated filtered seawater in a recirculation system equipped with a central filtering unit and UV unit. Tank water temperature, salinity, pH, and dissolved oxygen were continuously monitored and NH4+, NO3−, and NO2− were assessed every 7 days and maintained within optimal ranges. Fish were fed to apparent satiety twice a day (6 days per week) except for the 7 days prior to euthanization when they were fed only once. Three diets were formulated for this experiment (Sparos Lda., Loulé, Portugal; Table 1): a no-CHO control diet (CTRL) with no CHO (except for an inert filler of cellulose of no nutritional value to maintain pellet integrity in water); and two experimental diets, one with 33% DS, and another with 33% (RS). All diets were formulated to fulfill the known nutritional requirements of the species. Following the feeding period, each group was transferred to a 2H2O-enriched seawater tank for 6 days. This 200 l tank was maintained with an independent closed filtering system, but had similar characteristics as the other tanks used during the rearing phase in terms of size, opacity, filtering material, and water parameters. Seawater was enriched with 2H2O to 5% with the addition of 99% enriched 2H2O (Eurisotop, France), as previously described by Viegas et al. (19Viegas I. Mendes V.M. Leston S. Jarak I. Carvalho R.A. Pardal M.A. Manadas B. Jones J.G. Analysis of glucose metabolism in farmed European sea bass (Dicentrarchus labrax L.) using deuterated water.Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2011; 160: 341-347Crossref PubMed Scopus (27) Google Scholar), and 2H-enrichment was quantified after each set of experiments, as described by Jones, Merritt, and Malloy (20Jones J.G. Merritt M. Malloy C. Quantifying tracer levels of 2H2O enrichment from microliter amounts of plasma and urine by 2H NMR.Magn. Reson. Med. 2001; 45: 156-158Crossref PubMed Scopus (52) Google Scholar). During this 6 day period in 2H2O-enriched saltwater, fish were fed once a day and provided with the last meal 24 h before euthanization. Fish were anesthetized in a 30 l tank of 5% 2H-enriched saltwater containing 0.1 g l−1 of MS-222, measured, weighed, and sampled for blood from the caudal vein with heparinized syringes. Fish were then euthanized by cervical section; livers and samples of skeletal muscle and perivisceral fat were excised, weighed, freeze-clamped in liquid N2, and stored at −80°C until further analysis. All experimental procedures complied with the Guidelines of the European Union Council (86/609/EU).TABLE 1.Ingredients and proximate composition of the experimental diets provided to seabass (D. labrax)CTRLDSRSIngredients (%)FishmealaPeruvian fishmeal LT: 67% crude protein, 9% crude fat (EXALMAR, Peru).65.554.654.6Gelatinized pea starchbAquatex 8071, gelatinized dehulled microground pea meal: 23% crude protein, 50% starch (SOTEXPRO, France).—33.4—Raw pea starchcAquatex G2000 (bran), crude dehulled microground pea meal: 23% crude protein, 50% starch (SOTEXPRO, France).——33.4Fish oildMarine oil omega 3 (Henry Lamotte Oils GmbH, Germany).9.810.510.5Vitamin and mineral premixePVO 40.01 premix for marine fish (PREMIX Lda, Portugal).1.01.01.0Binder (diatomaceous earth)fKielseguhr (LIGRANA GmbH, Germany).0.50.50.5Cellulose (inert filler)gMicrocrystalline cellulose (Blanver, Brazil).23.2——Proximate composition (% dry weight)Dry matter96.095.697.4Crude protein50.250.250.2Crude fat16.116.116.1Starch0.217.817.8Ash11.59.311.1Gross energy (kJ g−1 dry weight)22.6622.0322.03a Peruvian fishmeal LT: 67% crude protein, 9% crude fat (EXALMAR, Peru).b Aquatex 8071, gelatinized dehulled microground pea meal: 23% crude protein, 50% starch (SOTEXPRO, France).c Aquatex G2000 (bran), crude dehulled microground pea meal: 23% crude protein, 50% starch (SOTEXPRO, France).d Marine oil omega 3 (Henry Lamotte Oils GmbH, Germany).e PVO 40.01 premix for marine fish (PREMIX Lda, Portugal).f Kielseguhr (LIGRANA GmbH, Germany).g Microcrystalline cellulose (Blanver, Brazil). Open table in a new tab A portion of liver was stored separately (n = 6 per diet) at −80°C for glycogen quantification, as described by Keppler and Decker (21Keppler D. Decker K. Bergmeyer H.U. Glycogen determination with amyloglucosidase.in: Peet R. Watts M. In Methods of Enzymatic Analysis. Academic Press Inc., New York1974: 1127-1131Google Scholar). Blood glucose, TG, and hepatic TG levels were quantified by using commercial kits (Cromatest; Linear Chemicals, Spain). Glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.43) liver activities were assayed as previously described (22Metón I. Mediavilla D. Caseras A. Cantó M. Fernández F. Baanante I.V. Effect of diet composition and ration size on key enzyme activities of glycolysis-gluconeogenesis, the pentose phosphate pathway and amino acid metabolism in liver of gilthead sea bream (Sparus aurata).Br. J. Nutr. 1999; 82: 223-232Crossref PubMed Google Scholar). All enzyme activity assays were carried out at 30°C and followed at 340 nm. Total protein content was determined by the Bradford method (Bio-Rad, Spain) at 30°C in liver crude extracts using BSA as a standard and followed at 600 nm. All assays for metabolites, enzyme activities, and total protein were adapted for automated measurement using a Cobas Mira S spectrophotometric analyzer (Hoffman-La Roche, Switzerland). Enzyme activities were expressed per milligram of soluble protein (specific activity). One unit of enzyme activity was defined as the amount of enzyme necessary to transform 1 βmol of substrate per minute. Hepatic lipids from the remaining pulverized livers were extracted by the method of Folch, Lees, and Stanley (23Folch J. Lees M. Stanley G.H.S. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). The TG fraction was separated from the rest of the lipids by column chromatography according to the modified procedure described by Hamilton and Comai (24Hamilton J.G. Comai K. Rapid separation of neutral lipids, free fatty acids and polar lipids using prepacked silica sep-Pak columns.Lipids. 1988; 23: 1146-1149Crossref PubMed Scopus (191) Google Scholar). Briefly, extracted lipids were dissolved in hexane/methyl-t-butyl-ether mixture (200:3, v/v) and applied to a silica gel prepacked column (Sigma prepacked 2 g Discovery DSC-Si SPE tubes). The column was eluted with the hexane/methyl-t-butyl-ether mixture (200:3, v/v) and the fractions containing TG evaporated to dryness. To determine the identity and the purity of collected lipid fractions, thin-layer chromatography was carried out on silica gel plates. A petroleum ether/diethyl ether/acetic acid (7:1:0.1, v/v/v) system was used as the mobile phase (TG Rf = 0.55). Lipid fractions were visualized by iodine vapors. Body water 2H-enrichments were determined from 10 βl aliquots of fish plasma by 2H NMR, as described by Jones, Merritt, and Malloy (20Jones J.G. Merritt M. Malloy C. Quantifying tracer levels of 2H2O enrichment from microliter amounts of plasma and urine by 2H NMR.Magn. Reson. Med. 2001; 45: 156-158Crossref PubMed Scopus (52) Google Scholar). Water content was assumed to be 92% of total plasma. Fully relaxed 1H NMR spectra of TG samples were obtained at 25°C with a 14.1 T Agilent 600 system equipped with a 3 mm broadband probe. Spectra were acquired with a 90° pulse and 8 s of recycling time (3 s of acquisition time and 5 s pulse delay). Proton-decoupled 2H NMR spectra were obtained under the same conditions with the observe coil tuned to 2H. Up to 5,400 scans were collected per sample, corresponding to a maximum of 12 h collection time. TG 2H-enrichments were quantified from the 1H and 2H NMR spectra by measuring the 1H and 2H intensities of selected signals relative to the 1H and 2H intensities of a pyrazine standard, as described previously by Duarte et al. (16Duarte J.A.G. Carvalho F. Pearson M. Horton J.D. Browning J.D. Jones J.G. Burgess S.C. A high-fat diet suppresses de novo lipogenesis and desaturation but not elongation and triglyceride synthesis in mice.J. Lipid Res. 2014; 55: 2541-2553Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). From this methodology, the fraction of hepatic TG-bound FAs (or fatty acyls) derived from DNL and the fraction of newly synthesized TG-bound glycerol (or glyceryl) over the 2H2O administration were estimated. Excess enrichments were calculated after systematic subtraction of the values with 0.012%, the mean background 2H-enrichment value reported for FAs isolated from salmon muscle (25Aursand M. Mabon F. Martin G.J. High-resolution 1H and 2H NMR spectroscopy of pure essential fatty acids for plants and animals.Magn. Reson. Chem. 1997; 35: S91-S100Crossref Scopus (31) Google Scholar). If the values were below zero, these were considered as 0.0 for fractional synthetic rate (FSR) calculation purposes. Spectra were processed by applying exponential line broadening (1H, 0.1 Hz; 2H. 1.0 Hz) and analyzed using the curve-fitting routine supplied with ACDLabs 1D NMR processor software 2.4. Data are presented as mean ± SEM. ANOVA was used to test significant differences between the three dietary treatments. A posteriori Tukey's multiple comparisons test was performed when significant differences were found. Differences were considered statistically significant at P < 0.05. The daily growth index, somatic indexes, and biochemical parameters determined are summarized in Table 2. CTRL and DS fish had similar growth rates and somatic indexes, while RS fish had significantly lower values for these parameters. The perivisceral fat index was significantly higher for DS compared with RS, but neither group was significantly different from CTRL. Surprisingly, neither DS nor RS feeding resulted in significant increases of glycemia and tissue glycogen levels compared with the CTRL diet. Indeed, plasma glucose levels for RS were significantly lower compared with CTRL, while DS fish had intermediate values that were not significantly different from either RS or CTRL. In contrast to glycemic status, the effect of the diets on plasma and hepatic TG levels were more clear-cut with DS showing a distinctly more lipidic profile compared with the other two diets. Plasma TG levels were significantly elevated in DS compared with both RS and CTRL, while hepatic TG was ∼3-fold higher for fish fed DS diet compared with both CTRL and RS. Activities of enzymes that can provide NADPH for lipogenesis were affected by the inclusion and type of CHO used (Fig. 1). DS-fed fish showed higher activity relative to CTRL in both enzymes, even if in the case of G6PDH, these differences were not statistically significant. RS-fed fish on the other hand, revealed lower activity compared with DS-fed fish for both enzymes.TABLE 2.Growth and physiological parameters for seabass (D. labrax) fed with a CTRL diet, a 30% DS diet, and a 30% RS dietCTRLDSRSGrowth parametersDaily growth indexaDaily growth index = [(final bodyweight1/3 − initial body weight1/3)/days] × 100.0.79 ± 0.03bHepatosomatic index = (liver weight/body weight) × 100.0.80 ± 0.02bHepatosomatic index = (liver weight/body weight) × 100.0.63 ± 0.04aDaily growth index = [(final bodyweight1/3 − initial body weight1/3)/days] × 100.Hepatosomatic indexbHepatosomatic index = (liver weight/body weight) × 100.1.56 ± 0.03bHepatosomatic index = (liver weight/body weight) × 100.1.50 ± 0.04bHepatosomatic index = (liver weight/body weight) × 100.1.01 ± 0.05aDaily growth index = [(final bodyweight1/3 − initial body weight1/3)/days] × 100.Perivisceral fat indexcPerivisceral fat index = (perivisceral fat weight/body weight) × 100.4.93 ± 0,24ab5.11 ± 0.18bHepatosomatic index = (liver weight/body weight) × 100.4.22 ± 0.21aDaily growth index = [(final bodyweight1/3 − initial body weight1/3)/days] × 100.PlasmaGlucose (mM)10.0 ± 1.5bHepatosomatic index = (liver weight/body weight) × 100.8.2 ± 0.5ab6.0 ± 0.7aDaily growth index = [(final bodyweight1/3 − initial body weight1/3)/days] × 100.TGs (mM)11.1 ± 1.6aDaily growth index = [(final bodyweight1/3 − initial body weight1/3)/days] × 100.18.2 ± 1.4bHepatosomatic index = (liver weight/body weight) × 100.9.9 ± 1.6aDaily growth index = [(final bodyweight1/3 − initial body weight1/3)/days] × 100.LiverGlycogen (g/100 g−1 liver)9.9 ± 0.3bHepatosomatic index = (liver weight/body weight) × 100.11.6 ± 0.6bHepatosomatic index = (liver weight/body weight) × 100.4.1 ± 1.1aDaily growth index = [(final bodyweight1/3 − initial body weight1/3)/days] × 100.TGs (g/100 g−1 liver)11.1 ± 0.2aDaily growth index = [(final bodyweight1/3 − initial body weight1/3)/days] × 100.35.4 ± 0.7bHepatosomatic index = (liver weight/body weight) × 100.8.7 ± 0.1aDaily growth index = [(final bodyweight1/3 − initial body weight1/3)/days] × 100.Mean values ± SEM are presented. Significant differences between dietary treatments are indicated by different letters (one-way ANOVA followed by Tukey's test).a Daily growth index = [(final bodyweight1/3 − initial body weight1/3)/days] × 100.b Hepatosomatic index = (liver weight/body weight) × 100.c Perivisceral fat index = (perivisceral fat weight/body weight) × 100. Open table in a new tab Mean values ± SEM are presented. Significant differences between dietary treatments are indicated by different letters (one-way ANOVA followed by Tukey's test). Following extraction and subsequent isolation from other lipid classes by solid phase extraction, hepatic TG gave well-characterized 1H NMR spectra (Fig. 2A, Table 3). The FA/glyceryl ratio was consistent and was ∼3 in all the diets, as would be expected from a successful TG separation (Table 4). Signals from saturated FA and MUFA moieties dominated the spectrum, while contributions from PUFAs were relatively minor, as seen by the resolved PUFA methyl resonance downfield of the main methyl signal. This was also reflected in the composition and structure of FAs calculated by 1H NMR (Table 4). Despite no significant differences observed in saturated FAs and unsaturated FAs between dietary treatments, the percentage of PUFAs increased from CTRL to RS. MUFAs revealed the inverse pattern with DS-fed fish presenting intermediate values in both cases. Consequently, the estimated FA chemical structure varied accordingly in terms of the average number of protons and methylene units.TABLE 3Functional groups (with NMR signal assignments in bold), chemical shifts, and positional excess of 2H-enrichments in hepatic TG of seabass (D. labrax) fed with a CTRL diet, a 30% DS diet, and a 30% RS diet after a 6 day residence in a tank with 5% 2H-enriched waterSignalFunctional GroupChemical Shift (ppm)Assignment2H-enrichmentsCTRLDSRSA+BNon n-3 + partial n-6 methyls0.80CH3-CH2-0.043 ± 0.010b0.030 ± 0.007b−0.004 ± 0.004aCn-3 Methyls0.90CH3-CH2-CH=———DAliphatic chain methylenes1.20CH3-(CH2)n-0.050 ± 0.011b0.023 ± 0.005ab−0.001 ± 0.002aEβ Methylenes1.50-CH2-CH2-COO-0.093 ± 0.013b0.033 ± 0.003a0.008 ± 0.006aFMU allylic hydrogens1.90-CH2-CH=CH-0.023 ± 0.0060.030 ± 0.0060.009 ± 0.008GPU allylic hydrogens2.00-CH2-CH=CH-———Hα Methylenes2.20-CH2-CH2-COO-0.094 ± 0.014b0.032 ± 0.005a0.010 ± 0.011aIDHA α and β methylenes2.30-CH2-CH2-COO-———J+KBisallylic methylenes2.70-CH=CH-CH2-CH=CH-———Lsn-1, sn-3 of TG-glycerol4.15HOCH2-CHOH-CH2OH0.661 ± 0.037b0.312 ± 0.020b0.306 ± 0.057bMsn-2 of TG-glycerol5.15HOCH2-CHOH-CH2OH———NOlefinic hydrogens5.25-CH=CH-———OChloroform7.25Solvent———PPyrazine8.60Standard———Mean values ± SEM are presented (n = 6). Significant differences between dietary treatments are indicated by different letters (one-way ANOVA followed by Tukey's test; P < 0.05). Enrichments adjusted for tank water at 5.0% 2H2O. MU, monounsaturated; PU: polyunsaturated. Open table in a new tab TABLE 4Percentage of lipid species and chemical structure as determined from 1H NMR spectra of hepatic TG of seabass (D. labrax) fed with a CTRL diet, a 30% DS diet, and a 30% RS dietCTRLDSRSLipid species (%)Non n-382.0 ± 2.078.6 ± 1.880,9 ± 6.9SFA26.1 ± 1.525.7 ± 2.523.8 ± 0.6UFA73.9 ± 1.574.3 ± 2.576.2 ± 0.6PUFA27.4 ± 1.7a33.9 ± 2.0b41.2 ± 0.6cMUFA46.5 ± 0.9c40.8 ± 0.8b31.8 ± 1.0aChemical structureANC18.8 ± 0.418.5 ± 0.417.1 ± 1.3ANP31.3 ± 0.5b29.9 ± 0.4ab28.1 ± 1.0aOlefinic (HC=CH)2.6 ± 0.33.0 ± 0.32.6 ± 0.9Methylenic (CH2)11.5 ± 0.3b10.1 ± 0.2a9.9 ± 0.4aFA/glyceryl2.9 ± 0.12.8 ± 0.12.7 ± 0.0AMW289.0 ± 5.6283.8 ± 5.4265.9 ± 16.9Mean values ± SEM are presented (n = 6). Significant differences between dietary treatments are indicated by different letters (one-way ANOVA followed by Tukey's test; P < 0.05). Chemical structure: FAs were considered polymers of olefinic (HC=CH) and methylenic (CH2) subunits [i.e., −OOC−(CH2)x−(HC=CH)y−CH3] calculated as in Duarte et al. (16Duarte J.A.G. Carvalho F. Pearson M. Horton J.D. Browning J.D. Jones J.G. Burgess S.C. A high-fat diet suppresses de novo lipogenesis and desaturation but not elongation and triglyceride synthesis in mice.J. Lipid Res. 2014; 55: 2541-2553Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) except for the olefinic hydrogens that were calculated directly as equal to the correspondent area in the spectra. ANC, average number of carbons; ANP, average number of protons; AMW, average molecular weight. SFA, saturated FA; UFA, unsaturated FA. Open table in a new tab Mean values ± SEM are presented (n = 6). Significant differences between dietary treatments are indicated by different letters (one-way ANOVA followed by Tukey's test; P < 0.05). Enrichments adjusted for tank water at 5.0% 2H2O. MU, monounsaturated; PU: polyunsaturated. Mean values ± SEM are presented (n = 6). Significant differences between dietary treatments are indicated by different letters (one-way ANOVA followed by Tukey's test; P < 0.05). Chemical structure: FAs were considered polymers of olefinic (HC=CH) and methylenic (CH2) subunits [i.e., −OOC−(CH2)x−(HC=CH)y−CH3] calculated as in Duarte et al. (16Duarte J.A.G. Carvalho F. Pearson M. Horton J.D. Browning J.D. Jones J.G. Burgess S.C. A high-fat diet suppresses de novo lipogenesis and desaturation but not elongation and triglyceride synthesis in mice.J. Lipid Res. 2014; 55: 2541-2553Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) except for the olefinic hydrogens that were calculated directly as equal to the correspondent area in the spectra. ANC, average number of carbons; ANP, average number of protons; AMW, average molecular weight. SFA, saturated FA; UFA, unsaturated FA. Because 1H and 2H signals are essentially isochronous, the identity of the 2H
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