Isomerization of stable isotopically labeled elaidic acid to cis and trans monoenes by ruminal microbes

A previous study showed that oleic acid was converted by mixed ruminal microbes to stearic acid and also converted to a multitude of trans octadecenoic acid isomers. This study traced the metabolism of one of these trans C18:1 isomers upon its incubation with mixed ruminal microbes. Unlabeled and labeled (18-[13C]trans-9 C18333305) elaidic acid were each added to four in vitro batch cultures with three cultures inoculated with mixed ruminal bacteria and one uninoculated culture. Samples were taken at 0, 12, 24, and 48 h and analyzed for 13C enrichment in component fatty acids by gas chromatography-mass spectrometry. At 0 h of incubation, enrichment was detected only in elaidic acid. By 48 h of incubation, 13C enrichment was 18% (P < 0.01) for stearic acid, 7% to 30% (P < 0.01) for all trans C18:1 isomers having double bonds between carbons six through 16, and 5% to 10% for cis-9 and cis-11 monoenes. After 48 h, 13C enrichment in the uninoculated cultures was only detected in the added elaidic acid.This study shows trans fatty acids exposed to active ruminal cultures are converted to stearic acid but also undergo enzymic isomerization yielding a multitude of positional and geometric isomers. A previous study showed that oleic acid was converted by mixed ruminal microbes to stearic acid and also converted to a multitude of trans octadecenoic acid isomers. This study traced the metabolism of one of these trans C18:1 isomers upon its incubation with mixed ruminal microbes. Unlabeled and labeled (18-[13C]trans-9 C18333305) elaidic acid were each added to four in vitro batch cultures with three cultures inoculated with mixed ruminal bacteria and one uninoculated culture. Samples were taken at 0, 12, 24, and 48 h and analyzed for 13C enrichment in component fatty acids by gas chromatography-mass spectrometry. At 0 h of incubation, enrichment was detected only in elaidic acid. By 48 h of incubation, 13C enrichment was 18% (P < 0.01) for stearic acid, 7% to 30% (P < 0.01) for all trans C18:1 isomers having double bonds between carbons six through 16, and 5% to 10% for cis-9 and cis-11 monoenes. After 48 h, 13C enrichment in the uninoculated cultures was only detected in the added elaidic acid. This study shows trans fatty acids exposed to active ruminal cultures are converted to stearic acid but also undergo enzymic isomerization yielding a multitude of positional and geometric isomers. Anaerobic bacteria that colonize the rumen, or largest of the four stomach compartments in ruminant species, carry on a process of lipid biohydrogenation whereby double bonds in unsaturated fatty acids are partially or completely eliminated. Linoleic acid disappeared completely by 50 h when incubated with mixed ruminal microorganisms (1Kellens M.J. Goderis H.L. Tobback P.P. Biohydrogenation of unsaturated fatty acids by a mixed culture of rumen microorganisms.Biotechnol. Bioeng. 1986; 28: 1268-1276Google Scholar). As linoleic acid disappeared, transient increases in a number of trans diene isomers were seen, followed by the accumulation of trans-11 C18:1. During the later hours of incubation, the trans-11 C18:1 declined slowly and was accompanied by an increase in stearic acid concentration (1Kellens M.J. Goderis H.L. Tobback P.P. Biohydrogenation of unsaturated fatty acids by a mixed culture of rumen microorganisms.Biotechnol. Bioeng. 1986; 28: 1268-1276Google Scholar). Oleic acid biohydrogenation is generally presented as a direct conversion to stearic acid without the formation of trans intermediates (1Kellens M.J. Goderis H.L. Tobback P.P. Biohydrogenation of unsaturated fatty acids by a mixed culture of rumen microorganisms.Biotechnol. Bioeng. 1986; 28: 1268-1276Google Scholar, 2Griinari J.M. Bauman D.E. Biosynthesis of conjugated linoleic acid and its incorporation into meat and milk in ruminants.in: Yurawecz M.P. Mossoba M.M. Kramer J.K.G. Pariza M.W. Nelson G.J. Advances in Conjugated Linoleic Acid Research. AOCS Press, Champaign, IL.1999: 180-200Google Scholar). When 13C-labeled oleic acid was incubated with ruminal microorganisms in a recent study (3Mosley E.E. Powell G.L. Riley M.B. Jenkins T.C. Microbial biohydrogenation of oleic acid to trans isomers in vitro.J. Lipid Res. 2002; 43: 290-296Google Scholar), enrichment was observed not only in stearic acid but also in all trans C18:1 isomers having double bonds at carbon positions six through 16. However, the fate of these positional isomers of trans-C18:1 is not clear. Trans-11 C18:1 is readily converted to stearic acid by select ruminal bacteria (4Jenkins T.C. Lipid metabolism in the rumen.J. Dairy Sci. 1993; 76: 3851-3863Google Scholar), but its conversion to other trans monenes has not been reported. Kemp et al. (5Kemp P. Lander D.J. Gunstone F.D. The hydrogenation of some cis- and trans-octadecenoic acids to stearic acid by a rumen Fusocillus sp.Br. J. Nutr. 1984; 52: 165-170Google Scholar) incubated cis (cis-2 and cis-4 to cis-13) and trans (trans-2 and trans-5 to trans-13) octadecenoic acid isomers with a rumen Fusocillus species. They wanted to test the ability of Fusocillus to hydrogenate the octadecenoic acids to stearic acid. Cis-5 to cis-13 and trans-5 to trans-13 isomers were all hydrogenated to some extent by late log-phase cultures incubated for 3 h. Between 73% and 79% of cis-5 to cis-11 isomers were converted to stearic acid. However, cis-12 (30%) and cis-13 (5%) were poorly hydrogenated. Of the trans isomers, 45% of trans-8, trans-9, and trans-10 were converted to stearic acid but other isomers were poorly hydrogenated. This study was conducted to determine the fate of carbons from trans-9 C18:1 (elaidic acid) following its incubation with mixed ruminal microbes for 48 h. Cultures of mixed ruminal microbes were supplemented with 13C-labeled elaidic acid to determine possible enrichment in stearic acid and other monene isomers. Labelled elaidic acid (18-[13C]trans-9 C18:1) was purchased from CDN Isotopes (Quebec, Canada). Unlabelled elaidic acid (99% pure) was purchased from Sigma-Aldrich Chemical Company (St. Louis, MO). All solvents were HPLC or GC grade. Dimethyl disulfide (DMDS), silver nitrate crystal, anhydrous ethyl ether, iodine, and sodium thiosulfate were purchased from Fisher Scientific (Pittsburgh, PA). Microbial conversion of elaidic acid was studied in cultures of mixed gut microbes taken from the stomach (rumen compartment) of cattle. Cultures were maintained in 125 ml Erlenmeyer flasks containing 500 mg of ground hay, 40 ml of media, 50 mg of elaidic acid, and 2 ml of reducing solution according to Goering and Van Soest (6Goering H.K. Van Soest P.J. Forage Fiber Analysis (Apparatus, Reagents, Procedures, and Some Applications). ARS-USDA, Washington, DC1970Google Scholar). Unlabelled cultures received 400 μl of elaidic acid in ethanol (125 mg/ml). The labeled cultures received 400 μl of an elaidic acid solution in ethanol (125 mg/ml) consisting of 50% 18-[13C]elaidic acid and 50% unlabelled elaidic acid. Cultures containing unlabeled or labeled elaidic acid (n = 4) were run at 39°C under anaerobic conditions. Three of the four labeled and unlabeled flasks were inoculated with microbes collected from the rumen of a fistulated Holstein cow. Contents from the rumen were thoroughly mixed by hand 2 h after the morning feeding, strained through two layers of cheesecloth, and added (10 ml) to culture flasks while gassing continuously with CO2. The remaining labeled and unlabelled flask received an additional 10 ml of media in place of the ruminal inocula. Duplicate samples (5 ml) were taken from each culture at 0 h, 12 h, 24 h, and 48 h and immediately frozen. The samples were freeze-dried and then methylated according to Kramer et al. (7Kramer J.K.G. Fellner V. Dugan M.E.R. Sauer F.D. Mossoba M.M. Yurawecz M.P. Evaluating acid and base catalysts in the methylation of milk and rumen fatty acids with special emphasis on conjugated dienes and total trans fatty acids.Lipids. 1997; 32: 1219-1228Google Scholar). When stored, all samples containing fatty acid methyl esters (FAME) were stored in an organic solvent at −15°C. The FAME samples from each incubation time were taken to dryness under a stream of nitrogen gas and then dissolved in 0.4 ml of methylene chloride. The FAME were separated into saturated, trans monoene, cis monoene, and diene fractions using a modified procedure of Christie (8Christie W.W. Silver ion chromatography using solid-phase extraction columns packed with bonded-sulfonic acid phase.J. Lipid Res. 1989; 30: 1471-1473Google Scholar). The modified procedure is as follows: an Isolute® SCX-2 (International Sorbent Technology, Mid Glamorgan, UK) solid phase extraction column (500 mg, 10 ml reservoir) was wrapped to the level of the top of the absorbent bed with aluminum foil. The column was preconditioned by elution with 2 ml of acetonitrile. A solution of 20 mg of silver nitrate in 0.25 ml acetonitrile-water 10:1 (v/v) was allowed to flow through the solid phase extraction column. The column was flushed with acetonitrile (5 ml), acetone (5 ml), and methylene chloride (10 ml). The FAME sample in 0.4 ml methylene chloride was divided equally between two columns for better resolution and washed onto the column in methylene chloride (0.2 ml). Saturated fatty acids were eluted with methylene chloride (5 ml). The monoene fraction was separated into trans monoenes and cis monoenes by washing with 0.5% acetone in methylene chloride (5 ml) and 10% acetone in methylene chloride (5 ml), respectively. Dienes were eluted with acetone (5 ml). All fractions were eluted by gravity. Corresponding fractions from the two columns were combined and taken to dryness under a stream of nitrogen gas. The FAME in the saturated and diene fractions were dissolved in 200 μl of hexane and analyzed by gas chromatography-mass spectrometry (GC-MS). DMDS adducts of the trans monoene and cis monoene fractions were prepared using a modified procedure of Yamamoto et al. (9Yamamoto K. Shibahara A. Nakayama T. Kajimoto G. Determination of double-bond positions in methylene-interrupted dienoic fatty acids by GC-MS as their dimethyl disulfide adducts.Chem. Phys. Lipids. 1991; 60: 39-50Google Scholar). The modified procedure is as follows: the FAME fractions were treated with 0.35 ml of DMDS and 100 μl of iodine solution (6% iodine w/v in diethyl ether). The reaction mixtures were shaken in a 37° C water bath for 1 h and then diluted with diethyl ether-hexane (3 ml; 1:1, v/v). Iodine was removed by shaking with 10% sodium thiosulfate (200 μl). The organic phase was removed and the solvent was evaporated under a stream of nitrogen gas. The residue was dissolved in 200 μl of hexane and analyzed immediately by GC-MS. When stored, samples were stored no longer than 2 days in hexane at −15°C. Analysis of the FAME in the saturated and diene fractions, and the DMDS derivatives in the trans and cis fractions were analyzed by GC-MS as described by Mosley et al. (3Mosley E.E. Powell G.L. Riley M.B. Jenkins T.C. Microbial biohydrogenation of oleic acid to trans isomers in vitro.J. Lipid Res. 2002; 43: 290-296Google Scholar). Additional FAME samples containing 1 mg of C17:0 internal standard were analyzed on a gas chromatograph (Shimadzu GC-14A; Columbia, Maryland) equipped with a flame ionization detector and a 100 m × 0.25 mm, with 0.2 μm film capillary column coated with CP-Sil 88 (Chrompack, Raritan, New Jersey). The injector and detector temperatures were both 250°C. The carrier gas was H2 (33 cm/s) with an inlet pressure of 250 kPa. The column temperature was isothermal at 160°C (held for 45 min) to separate major fatty acids. The DMDS derivatives of FAME produce two distinctive spectral fragments that are indicative of the double bond position when analyzed by mass spectrometry. The F fragment is the methyl thio adduct of the methyl end of the FAME. The G fragment is the methyl thio adduct of the carboxyl end of the FAME. The atom percent excess (APE) was calculated from the mass abundance of the F and F + 1 fragments using the equation APE = (F + 1)/[F + (F + 1)]. In order to correct for the natural levels of 13C, the average APE of unlabeled cultures was subtracted from the APE of labeled cultures. Therefore, enrichment of the fatty acid with 13C was calculated as (APE labeled − average APE unlabeled)*100. Changes in fatty acid concentration (mg/5 ml culture) over time were determined by analysis of variance using the PROC GLM (general linear model) procedure of SAS (SAS Institute, Inc., Cary, NC). Means and standard deviations were determined by the PROC MEAN procedure of SAS, with enrichment analyzed by Student's t-test to determine if they differed from zero. Total fatty acid concentration in the cultures increased (P < 0.05) from 0 h to 12 h of incubation (4.80 to 5.23 mg/5 ml) then remained constant through 48 h (Fig. 1). The slight increase in total fatty acids over time is due to the lack of fatty acid catabolism by ruminal anaerobes combined with their ability to synthesize long-chain fatty acids de novo from fermentation acids (4Jenkins T.C. Lipid metabolism in the rumen.J. Dairy Sci. 1993; 76: 3851-3863Google Scholar). The concentration of elaidic acid declined (P < 0.05) over time. Unsaturated fatty acids exposed to ruminal microbes generally decrease in concentration due to their biohydrogenation to more saturated end products. Stearic acid concentration increased (P < 0.05) over time, but the concentrations of C16:0 and cis-9 C18:1 were small at 0 h and changed little. The enrichment of C18:0 at 12 h through 48 h (Fig. 2)supports biohydrogenation as the process for the conversion of elaidic acid to C18:0, and accounts for the disappearance of trans-9 C18:1 over time. Earlier work (5Kemp P. Lander D.J. Gunstone F.D. The hydrogenation of some cis- and trans-octadecenoic acids to stearic acid by a rumen Fusocillus sp.Br. J. Nutr. 1984; 52: 165-170Google Scholar) also showed the conversion of elaidic acid to C18:0 by a ruminal Fusocillus species. Mosley et al. (3Mosley E.E. Powell G.L. Riley M.B. Jenkins T.C. Microbial biohydrogenation of oleic acid to trans isomers in vitro.J. Lipid Res. 2002; 43: 290-296Google Scholar) recently confirmed that carbons from oleic acid were transferred to stearic acid plus a number of positional isomers of trans monoenes in cultures of mixed ruminal microbes. This study extended those observations by showing that one of those positional isomers, namely trans-9 C18:1, was converted to stearic acid. When the results of the two investigations are taken together, they suggest a biohydrogenation pathway for oleic acid that is more similar to linoleic acid than is usually stated. Linoleic acid is acted upon by an isomerase yielding several trans conjugated dienes, which in turn are reduced to trans monoene intermediates with trans-11 C18:1 being the most abundant (4Jenkins T.C. Lipid metabolism in the rumen.J. Dairy Sci. 1993; 76: 3851-3863Google Scholar). Conversely, the biohydrogenation of oleic acid, as it is usually depicted, proceeds directly to stearic acid without the action of an isomerase or the accumulation of trans monenes. The results of the current study and the previous study by Mosley et al. (3Mosley E.E. Powell G.L. Riley M.B. Jenkins T.C. Microbial biohydrogenation of oleic acid to trans isomers in vitro.J. Lipid Res. 2002; 43: 290-296Google Scholar) indicate the presence of one or more isomerases that convert oleic acid to many trans monenes, which then undergo reduction to stearic acid. The enrichments were not different (P > 0.05) from zero for C16:0 or C18:2 (Fig. 2). The lack of 13C-label in C16:0 rules out degradation of elaidic acid to shorter acyl chains or acetate and then utilization of the labeled acetate for elongation or even de novo synthesis of C16:0. It also rules out biohydrogenation of elaidic acid to C18:0 and then chain shortening of the C18:0 to C16:0. The lack of enrichment in C18:2 is consistent with the inability of anaerobes to synthesize polyunsaturated fatty acids. The synthesis of polyunsaturated fatty acids is restricted to aerobes via the oxygen-requiring desaturation of previously formed saturated fatty acids (10Keweloh H. Heipieper H.J. Trans unsaturated fatty acids in bacteria.Lipids. 1996; 31: 129-137Google Scholar). Unexpectedly, the enrichment data showed conversion of elaidic acid to two cis isomers, cis-9 and cis-11 C18:1. The interconversion of geometric isomers by isomerization has been demonstrated in several bacterial species. Usually, cis to trans isomerization is described more frequently than trans to cis. Bacterial species will convert oleic acid to elaidic acid to invoke changes in membrane permeability that protect them from a variety of growth inhibitors, including toxicants (10Keweloh H. Heipieper H.J. Trans unsaturated fatty acids in bacteria.Lipids. 1996; 31: 129-137Google Scholar) or changes in ambient temperature (11Okuyama H. Okajima N. Sasaki S. Higashi S. Murata N. The cis-trans isomerization of the double bond of a fatty acid as a strategy for adaptation to changes in ambient temperature in the psychophilic bacterium vibrio-sp strain abe-1.Biochim. Biophys. Acta. 1991; 1084: 13-20Google Scholar). Kemp et al. (5Kemp P. Lander D.J. Gunstone F.D. The hydrogenation of some cis- and trans-octadecenoic acids to stearic acid by a rumen Fusocillus sp.Br. J. Nutr. 1984; 52: 165-170Google Scholar) reported cis/trans isomerizations in both directions by a ruminal bacterium, although cis to trans was more extensive than trans to cis. The high initial concentration of elaidic acid in this study may have promoted abnormally high trans to cis isomerization. The percentage of oleic acid originating from elaidic acid can be estimated by dividing enrichment for oleic acid at 48 h (6.4 ± 0.87%) by the average for elaidic acid at 0 h (36.4 ± as described by Mosley et al. (3Mosley E.E. Powell G.L. Riley M.B. Jenkins T.C. Microbial biohydrogenation of oleic acid to trans isomers in vitro.J. Lipid Res. 2002; 43: 290-296Google Scholar). This that of the oleic acid in the cultures from elaidic acid and possible trans to cis isomerization. of oleic acid in the cultures were from the bacterial and the It is possible to the and constant for of the cis double bonds to trans in the of formation of is and for it is of and Chemical Scholar). The in these is the for the conversion of a cis double bond to a trans double bond and is the cis double bond is as The constant for cis to trans isomerization estimated from the at is these isomers, under the at are for the cis and trans double bonds in elaidic and oleic the constant only the trans with an constant to The of positional isomerization of the double bonds for the monoenes are also to be The of trans to cis isomerization partially for the of oleic acid biohydrogenation that are reported. Biohydrogenation was 10 to percentage for oleic acid than for linoleic acid in with of different of polyunsaturated fatty acids on and fatty acid in J. Sci. Scholar, K. biohydrogenation of polyunsaturated fatty acids and their on and in Sci. Scholar) and Biohydrogenation and of chain fatty acids in on different of Sci. Scholar) a variety of In rumen in vitro of biohydrogenation also were for oleic acid with linoleic acid Jenkins T.C. P.J. of and of on the of and biohydrogenation of fatty acids in ruminal Dairy Sci. Scholar). However, if their data is for the 18% trans to cis isomerization observed in this oleic acid biohydrogenation increases four to six percentage not to the between oleic and linoleic acids. Kemp et al. (5Kemp P. Lander D.J. Gunstone F.D. The hydrogenation of some cis- and trans-octadecenoic acids to stearic acid by a rumen Fusocillus sp.Br. J. Nutr. 1984; 52: 165-170Google Scholar) out that cis/trans isomerization may not all from enzymic In their incubation of media for 24 h to some cis to trans isomerization. The was in this elaidic was not in oleic acid or other fatty acid in the cultures. the presence of the bacterial was for which an However, the of other in the as reducing as the of isomerization be elaidic acid also was converted to a number of other C18:1 trans positional isomers. At 0 h of incubation, enrichment was only detected in the elaidic acid added to the cultures (Fig. At 12 h, enrichment was in stearic acid C18:1 cis-9 C18:1 cis-11 C18:1 elaidic acid and trans-11 C18:1 At 24 h, enrichment was in stearic acid cis-9 C18:1 cis-11 C18:1 C18:1 C18:1 elaidic acid and trans-11 C18:1 enrichment for these isomers were similar for 12 h, 24 h, and 48 h, only 48 h enrichment data are in 3 for of with mixed ruminal microbes also to enrichment of a multitude of trans-C18:1 positional isomers (3Mosley E.E. Powell G.L. Riley M.B. Jenkins T.C. Microbial biohydrogenation of oleic acid to trans isomers in vitro.J. Lipid Res. 2002; 43: 290-296Google Scholar). these Mosley et al. (3Mosley E.E. Powell G.L. Riley M.B. Jenkins T.C. Microbial biohydrogenation of oleic acid to trans isomers in vitro.J. Lipid Res. 2002; 43: 290-296Google Scholar) the of one or more isomerases that a multitude of trans-C18:1 isomers. The results of this study that a trans-C18:1 is as the elaidic acid, it can be converted to many other positional isomers. Therefore, biohydrogenation of oleic acid by ruminal microbes produce a as trans-9 or trans-11 C18:1, followed by the isomerization of this to many other positional isomers. of the the end is the oleic acid is converted to a number of trans-C18:1 positional isomers when it is exposed to cultures of mixed ruminal microbes. As the of trans C18:1 isomers, it more to their a of the trans-11 C18:1 by ruminal microbes is converted to trans-11 C18:2 by a acid is to conjugated linoleic acid in Nutr. Scholar). The trans-11 as acid, has been as a and of G. J. Conjugated linoleic in and with special to J. Lipid Sci. Scholar). Changes in of ruminant can the of trans monoenes in the at the of biohydrogenation increased trans-10 C18:1 at the of the trans-11 C18:1 J.M. Bauman D.E. Biosynthesis of conjugated linoleic acid and its incorporation into meat and milk in ruminants.in: Yurawecz M.P. Mossoba M.M. Kramer J.K.G. Pariza M.W. Nelson G.J. Advances in Conjugated Linoleic Acid Research. AOCS Press, Champaign, IL.1999: 180-200Google Scholar). The the by the number is extended to Riley for with GC-MS atom percent excess dimethyl disulfide fatty acid methyl gas chromatography-mass spectrometry

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