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Dietary unsaturated fatty acids are extensively hydrogenated in the rumen, resulting in the formation of numerous intermediates that may exert physiological effects and alter the fat composition of ruminant-derived foods. A batch culture method was used to characterize the hydrogenation of linoleic acid (LeA) by strained rumen fluid in vitro. Incubations (n = 5) were performed in 100-mL flasks maintained at 39°C containing 400 mg of grass hay, 50 mL of buffered rumen fluid, and incremental amounts of LeA (0, 1.0, 2.5, 5.0, or 10.0 mg) for 0, 1.5, 3.0, 4.5, 6.0, and 9.0 h. The fatty acid composition of flask contents was determined using complimentary silver-ion thin-layer chromatography, gas chromatography mass-spectrometry, and silver-ion high-performance liquid chromatography. Linoleic acid was extensively (98.1, 97.6, 98.0, and 89.8% for additions of 1.0, 2.5, 5.0, and 10.0 mg of LeA, respectively) hydrogenated over time. Complete reduction of LeA to 18:0 was inhibited in direct relation to the amount of added substrate, the extent of which was greatest for the highest amount of LeA addition. Recoveries of 1.0, 2.5, 5.0, and 10.0 mg of added LeA as 18:0 averaged 73.6, 65.0, 57.3, and 10.7%, respectively. Incubation of incremental amounts of LeA resulted in a time-dependent accumulation of geometric isomers of 9,11 and 10,12 conjugated linoleic acid, several nonconjugated 18:2 isomers, and a wide range of cis 18:1 and trans 18:1 intermediates. Several unusual intermediates including cis-6,cis-12 18:2; cis-7,cis-12 18:2; and cis-8,cis-12 18:2, were found to accumulate in direct relation to the amount of added LeA, providing the first indications that hydrogenation of LeA by ruminal bacteria may also involve mechanisms other than hydrogen abstraction or isomerization of the cis-12 double bond. Fitting of single-pool, first-order kinetic models to experimental data indicated that the rate of LeA disappearance decreased with increases in substrate availability. Reduction of 18:1 and 18:2 intermediates occurred at much lower rates compared with conjugated linoleic acid and nonconjugated 18:2 isomer formation. In conclusion, the extent of LeA biohydrogenation in vitro was shown to be time- and dose-dependent with evidence that LeA is hydrogenated by ruminal bacteria via several distinct metabolic pathways. The accumulation of several unusual 18:2 isomers indicates that biohydrogenation of LeA also proceeds via mechanisms other than isomerization of the cis-12 double bond.
Characterizing the formation of specific biohydrogenation metabolites in the rumen is important with respect to understanding the metabolic origins of trans fatty acids in ruminant-derived foods and also the mechanisms underlying physiological responses to lipids in the diet of growing and lactating ruminants. On entering the rumen, dietary lipids are exposed to microbial lipases and the NEFA liberated are subject to biohydrogenation. Bacteria, rather than protozoa, are thought to be responsible, but relatively few strains capable of biohydrogenation have been identified (
Disappearance of LeA and formation of fatty acid intermediates over time during incubations with strained rumen fluid was determined to provide a detailed and comprehensive assessment of LeA metabolism in vitro. Intermediates formed during incubations with incremental amounts of LeA were identified and quantified using complimentary argentation silver-ion thin-layer chromatography (Ag+-TLC), silver-ion HPLC (Ag+-HPLC), gas chromatography (GC), and GC-MS analysis of fatty acid methyl esters (FAME) and GC-MS analysis of corresponding 4,4-dimethyloxazoline (DMOX) derivatives.
Materials and Methods
In Vitro Incubations
Linoleic acid (cis-9,cis-12 18:2) obtained from a commercial source (Larodan Fine Chemicals AB, Malmö, Sweden) was incubated as an oil-in-water suspension (
). Four stock solutions of LeA suspensions containing 0.4, 1.0, 2.0, and 4.0 mg of LeA/mL were prepared daily, immediately before incubations with strained rumen fluid. Suspensions were prepared by mixing deionized water (270, 665, 1,330, and 2,660 μL) with Tween 80 (10.0, 25.0, 50.0, and 100 mg) and LeA (8.0, 20.0, 40.0, and 80.0 mg), resulting in a final LeA concentration of 0.4, 1.0, 2.0, and 4.0 mg of LeA/mL. Linoleic acid was dispersed by repeated flushing with a pipette, followed by the dropwise addition of 2M NaOH (1, 3, 6, and 12 drops, respectively) to each stock solution and gentle shaking until all solutions were clear. Once prepared, suspensions of LeA were transferred to 20-mL volumetric flasks and diluted with deionized water. Amounts of LeA, Tween 80, and NaOH added to each flask were adjusted to be in same ratio for all incubations.
Rumen fluid was sampled from 1 nonlactating Finnish Ayrshire cow fitted with a rumen cannula and fed 8.7 kg of DM/d of a diet (forage:concentrate ratio 70:30, on a DM basis) comprising timothy-meadow fescue silage supplemented with a standard commercial compound feed (Suomen Rehu Oy, Espoo, Finland), declared as containing 166, 76, and 40 g/kg of DM of CP, crude fiber, and ether extract, respectively, for 12 wk before sample collection.
One and a half liters of rumen fluid was collected from the donor cow using a vacuum pump 1 h after feeding and transported in a thermos flask to the in vitro laboratory within 10 min. On arrival, rumen fluid was strained through 2 layers of cheesecloth to retain small particles, and mixed (1:2, vol/vol) with warm (39°C), degassed (CO2) modified McDougall's buffer. Preliminary investigations in our laboratory confirmed that this procedure ensures that small particles are retained in the rumen inoculum. Buffer was prepared on each day of incubation by weighing 9.3 g of NaHCO3 and 9.8 g of NaHPO4 into a volumetric flask, followed by the addition of 20 mL of chloride solution (prepared by diluting 0.47 g of NaCl, 0.57 g of KCl, 0.12 g of MgCl2, and 0.04 g of CaCl2 in deionized water to a final volume of 20 mL) and deionized water to a final volume of 1.5 L. Buffered strained ruminal fluid was degassed with CO2 for 10 min.
Fifty milliliters of buffered ruminal fluid; 400 mg of finely ground dry hay; 2.5 mL of LeA stock emulsions providing 1, 2.5, 5.0, or 10.0 mg of LeA; and 5 mg of nonadecanoic acid (Larodan Fine Chemicals AB) as an internal standard (19:0, 5 mg/mL in ethanol) were transferred into 100-mL glass flasks. Ground hay was included as a fermentation substrate, because anaerobes do not generate ATP from LeA (
). Flasks, containing rumen fluid, buffer, ground hay, and internal standard (19:0) were also prepared and used as blank samples. Each flask was degassed with CO2, covered with a tight-fitting butyl rubber stopper, and incubated in on a benchtop shaker (Infors HT AG, Bottmingen, Switzerland), operated at a rotation speed of 85 rpm and maintained in the dark at 39°C for 0, 1.5, 3.0, 4.5, 6.0, and 9.0 h.
At the end of each designated time point, incubations were stopped by placing the flasks into ice-cold water and the pH of flask contents was measured. The contents of each flask were frozen (−20°C), freeze-dried (B. Brown Christ Gamma 2–20; B. Braun Melsungen AG, Melsungen, Germany), weighed, and stored at −20°C until submitted for fatty acid determinations. Incubations were repeated over 5 separate days with 1 flask for each incubation time per treatment.
Fatty Acid Analysis
Deionized water (0.5 mL) was added to the 100 mg of freeze-dried flask contents. After adjusting the pH to 2.0 with 2 M hydrochloric acid, lipid was extracted using 4 mL of a mixture (3:2; vol/vol) of hexane and isopropanol (
). Lipid extraction was repeated using the same solvent mixture and both organic phases recovered were combined, washed with deionized water, dried over approximately 200 mg of sodium sulfate, and evaporated to dryness at 45°C under a constant stream of nitrogen. Thereafter, NEFA were separated from other lipid fractions by solid-phase extraction using aminopropyl-bonded silica cartridges (1 g, Mega Bond Elut, Varian sample preparation products; Varian Inc., Harbor City, CA). Cartridges were preconditioned at atmospheric pressure with hexane and samples were allowed to penetrate the solid phase before the elution of neutral lipids with hexane and isopropanol (3:1; vol/vol). Nonesterified fatty acids were liberated using a mixture of acetic acid and diethyl ether (2:98 vol.vol) and the column effluent was evaporated to dryness under nitrogen at 45°C. Methyl esters were prepared by incubation of NEFA with methanolic sulfuric acid (1% vol/vol) at 50°C for 30 min (
). The reaction mixture was washed with aqueous sodium chloride (5% wt/vol) to promote the recovery of FAME in hexane. After washing with aqueous sodium hydrogen carbonate (2% wt/vol) the hexane-soluble phase was dried with sodium sulfate and transferred to glass GC vials.
Fatty acid methyl esters were separated and quantitated using a gas chromatograph (5890, Series II; Hewlett-Packard, Wilmington, DE) equipped with a flame-ionization detector, automatic injector, and a 100-m fused silica capillary column (i.d. 0.25 mm) coated with a 0.2-μm film of cyanopropyl polysiloxane (CP-SIL 88, Chrompack 7489; Chrompack International BV, Middelburg, the Netherlands). The total FAME profile in a 1-μL sample volume at a split ratio of 1:70 was determined using a temperature gradient program with helium as the carrier gas operated at constant pressure (195 kPa) at a flow rate of 0.6 mL/min. Following sample injection, the column temperature was maintained at 70°C for 1 min, increased at a rate of 30°C/min to 170°C, held for 54 min and raised to 220°C at a rate of 30°C/min and maintained at 220°C for 15 min. The total run time was 75 min. Injector and detector temperatures were maintained at 260 and 240°C, respectively.
Peaks were identified based on retention time comparisons with authentic FAME standards (GLC #67 and GLC#74; Nu-Check-Prep Inc., Elysian, MN). Methyl esters not contained in commercially available standards were formally identified based on GC-MS analysis of DMOX derivatives prepared from FAME fractionated according to the degree of unsaturation and configuration of the double bonds by Ag+-TLC. The TLC plates (Silica gel G, 200 × 200 mm and 0.50-mm thickness; No. 1.13894; Merck Co., Darmstadt, Germany) were impregnated with Ag+ by immersion in a silver nitrate solution (5%, wt/vol in acetonitrile) for 20 min. Following the application of approximately 15 mg of lipid, plates were developed with a mixture of hexane and diethyl ether (80:20; vol/vol) and visualized under UV light at 302 nm (UV trans-illuminator, 2011 Macrovue; LKB, Bromma, Sweden) after spraying with a 0.2% (wt/vol) solution of 2′,7′-dichlorofluorescein in methanol (
Methods for analysis of conjugated linoleic acids and trans-18:1 isomers in dairy fats by using a combination of gas chromatography, silver-ion thin-layer chromatography/gas chromatography, and silver-ion liquid chromatography.
). Five separated bands were individually scrapped off the plates and transferred to clean test tubes. A mixture of geometric isomers of 9,12 18:2 (L-8404; Sigma, St. Louis, MO) of known composition was used to assign the geometry of double bonds for fatty acids recovered in each fraction. Methyl esters were recovered from the silica after the addition of 4 mL of methanol, 8 mL of hexane, and 4 mL of a 6% (wt/vol) solution of aqueous sodium chloride (
Identification of minor fatty acids and various nonmethylene-interrupted diene isomers in mantle, muscle, and viscera of the marine bivalve Megangulus zyonoensis.
). After drying over anhydrous sodium sulfate, the hexane soluble phase was evaporated to dryness under nitrogen at room temperature and used to prepare DMOX derivatives.
The DMOX derivatives were prepared from FAME by incubation overnight (18 h) with 250 mg of 2-amino, 2-methyl-1-propanol under a nitrogen atmosphere according to
, with the exception that a temperature of 170°C was used. Once cooled, the reaction mixture was dissolved with 5 mL of a mixture of diethyl ether and hexane (1:1, vol/vol) and washed with 5 mL of deionized water. Organic extracts were combined, washed with 3 mL of deionized water, dried over anhydrous sodium sulfate, and evaporated to dryness under nitrogen at room temperature. The DMOX derivatives were dissolved in hexane and analyzed using a gas chromatograph (6890; Hewlett-Packard) equipped with a 100-m CP-Sil 88 fused silica capillary column (Chrompack International BV) and selective quadrupole mass detector (model 5973N; Agilent Technologies Inc., Wilmington, DE) connected to a dedicated computer with ChemStation software installed. Total DMOX profiles in a 4-μL sample volume were determined using helium as the carrier gas and the same temperature gradient applied during GC analysis of FAME. The mass spectrometer was operated at 230°C in the electron impact ionization mode and mass spectra were recorded under the ionization energy of 70 eV. Both the ion source and interface temperatures were maintained at 230°C.
Interpretation of mass spectra was undertaken according to published guidelines (
Structure analysis of fatty acids by gas chromatography-low resolution electron impact mass spectrometry of their 4, 4-dimethyloxazoline derivatives—A review.
), with an interval of 12 amu between the most intense peaks of clusters of ions containing n and n – 1 carbon atoms being interpreted as cleavage of the double bond between carbon n and n + 1 in the fatty acid moiety. In addition, odd-numbered fragments at m/z 139, 153, and 167 accompanied by even mass ions at m/z 138, 152, and 166 in the mass spectrum of DMOX derivatives were used as diagnostic ions to locate double bonds at Δ4, Δ5, and Δ6, respectively (
Structure analysis of fatty acids by gas chromatography-low resolution electron impact mass spectrometry of their 4, 4-dimethyloxazoline derivatives—A review.
). When available, the deduced fatty acid structure was verified by comparison with an online reference spectra library (http://lipidlibrary.aocs.org/ms/masspec.html). Double-bond geometry was deduced based on the relative abundance of FAME in each band recovered by Ag+-TLC, retention time during GC analysis, and the elution order of geometric isomers of 9,12 18:2 methyl esters.
The distribution of CLA isomers formed during in vitro incubations was determined using an HPLC system (Model 1090; Hewlett-Packard) equipped with 4 silver impregnated silica columns (ChromSpher 5 Lipids, 250 × 4.6 mm; 5-μm particle size; Varian Ltd., Walton-on-Thames, UK) coupled in series. Methyl esters of CLA were separated under isocratic conditions at 22°C using 0.1% (vol/vol) acetonitrile in heptane at a flow rate of 1 mL/min and monitoring column effluent at 233 nm (
). Typical injection volumes were 10 to 20 μL, representing <250 μg of lipid. Identification of CLA isomers was performed using commercially available CLA methyl ester standards (Matreya Inc., Pleasant Gap, PA; Sigma) and was verified by cross-referencing with the elution order reported in the literature (
Relative retention order of all isomers of cis/trans conjugated linoleic acid FAME from the 6,8- to 13,15-positions using silver ion HPLC with two elution systems.
) using cis-9,trans-11 CLA as a landmark isomer. Concentrations of specific conjugated isomers were calculated based on proportionate peak area responses determined by HPLC and the sum of trans-7,cis-9 CLA; trans-8,cis-10 CLA; and cis-9,trans-11 CLA determined as a single peak during GC analysis.
Calculations and Statistical Analysis
Amounts of fatty acids expressed as milligrams per flask were determined based on measurements of fatty acid composition and the fatty acid content of incubation residues determined using nonadecanoic acid as an internal standard. Concentrations of fatty acids were corrected by subtracting the amount of fatty acids measured in corresponding blank samples containing rumen fluid and all reagents except LeA from the amounts of fatty acids determined for all treatment samples. Corrected values were used to calculate LeA disappearance and the rates of biohydrogenation intermediate and end-product formation. Kinetic parameters of LeA disappearance expressed as a percentage of the initial amount added were estimated according to a first-order exponential model (
where Qt represents the percentage of LeA that disappears in time t (h), expressed as a percentage of the initial amount; a is the disappearance of LeA at time 0 h (expressed as a percentage of the initial amount); b is the percentage of LeA that can potentially disappear during incubations with ruminal fluid (expressed as the percentage of the initial amount); and c is the fractional rate of fraction b (expressed as 1/h). Parameters a, b, and c for LeA inclusion levels of 1.0, 2.5, and 5.0 mg/flask and for each day of incubation were computed using the NLIN procedure of the SAS (version 9.2; SAS Institute Inc., Cary, NC). Disappearance of LeA over time at the highest level of inclusion (10 mg/flask) was found to be linear and, therefore, the parameters to describe the kinetics of LeA biohydrogenation were evaluated by linear regression.
Measurements of the amount of biohydrogenation intermediates and 18:0 formed over time during incubations of incremental amounts of LeA with rumen fluid for each day of incubation were averaged to generate mean values for each incubation time per treatment for each day of incubation. These data were used to estimate fractional rates of transfer among fatty acid pools using WinSAAM software (Version 3.0.7; New Bolton Center, Biostatistics Unit, University of Pennsylvania, Philadelphia; available at http://www.winsaam.com) according to the model outlined by
. Representation of fatty acid pools used to estimate fractional rates of LeA biohydrogenation is shown in Figure 1. Intermediates formed during the initial biohydrogenation of LeA were assigned to 1 of 2 pools; CLA and nonconjugated (NC) 18:2, both of which were assumed to be reduced to 18:1 intermediates. Isomers of CLA are known to be reduced to 18:1 isomers during incubations with ruminal bacteria (
). Therefore, all 18:1 products were considered as a single fatty acid pool (Figure 1).
Figure 1Model of biohydrogenation of linoleic acid (LeA) during incubations with strained ruminal fluid. Boxes represent fatty acid pools and arrows indicate the transfer of fatty acids between pools during biohydrogenation. Linoleic acid, geometric isomers of conjugated linoleic acid (CLA), nonconjugated 18:2 isomers (NC 18:2), cis and trans 18:1 intermediates (18:1), and stearic acid (18:0).
Data on the incubation pH, rates of LeA disappearance, fractional rates, and amounts of intermediates that accumulated over time during incubations of LeA with strained ruminal fluid were analyzed by ANOVA for repeated measures using the mixed linear model procedure of SAS with a statistical model that included the effects of LeA addition, incubation time, and their interaction, with incubation day as the repeated variable assuming a compound symmetry covariance structure fitted on the basis of Akaike information and Schwarz Bayesian model-fit criteria. Sums of squares for treatment effects were further separated using polynomial contrasts into single degree-of-freedom comparisons to test for the significance of linear, quadratic, and cubic components of the effects of LeA addition on disappearance rates, accumulation of intermediates, and appearance of 18:0 end product. Least squares means are reported and treatment effects declared significant at P < 0.05.
Results
pH
During the course of incubations, the pH of fermentation flask contents decreased from 6.6 to 6.2. The pH of incubation flasks was not altered by the initial amount of LeA (P > 0.05) or due to interactions between the amount of added LeA and incubation time (P > 0.05; Figure 2).
Figure 2Temporal changes in mean pH of flask contents during incubations of 1.0 (●), 2.5 (○), 5.0 (▴), and 10.0 (▵) mg of linoleic acid with strained ruminal fluid. Values represent least squares means (n = 5; SEM = 0.018).
Disappearance of LeA, Accumulation of Biohydrogenation Intermediates, and Formation of Stearic Acid
Incubations with strained ruminal fluid resulted in extensive biohydrogenation of LeA. At the end of 9 h incubations, 98.1, 97.6, 98.0, and 89.8%, respectively, of 1.0, 2.5, 5.0, and 10.0 mg of added LeA disappeared. A significant amount of LeA was found to disappear several minutes after the start of incubations with strained ruminal fluid, but the proportion of LeA disappearing at time zero decreased linearly (P < 0.05) with LeA addition (Table 1). Furthermore, increases in LeA addition enhanced linearly (P = 0.001) the percentage of potential LeA disappearance from 71.2 to 87.4% but decreased the fractional rate of this fraction from 54.1 to 37.5%/h (Table 1). At the highest level of substrate addition, the rate of LeA disappearance was found to be constant at 7.6%/h over the 9-h incubation period (Figure 3). The sum of LeA, 18-carbon unsaturated biohydrogenation intermediates, and 18:0 averaged 1.15, 2.50, 4.97, and 10.08 mg/flask over the course of 0 to 9-h incubations with 1.0, 2.5, 5.0, and 10.0 mg of added LA, respectively. Incubations of incremental amounts of LeA resulted in a dose- and time-dependent accumulation of 18:2 isomers, 18:1 intermediates, and 18:0 (Table 2; Figure 4). Production of 18:0 increased in response to LeA addition, but at the highest level, complete biohydrogenation of LeA to 18:0 decreased (Figure 4). Percentages of the initial amount of LeA incubated recovered as 18:0 after 9-h incubations were 73.6, 65.0, 57.3, and 10.7% for 1.0, 2.5, 5.0, and 10.0 mg of added LeA, respectively. Decreases in the complete hydrogenation of LeA were associated with linear or quadratic increases (P < 0.001) in the accumulation of CLA, NC 18:2, and 18:1 intermediates (Table 2). Increasing the amounts of LeA incubated with strained ruminal fluid resulted in a linear (P < 0.001) increase in the ratio of total NC 18:2:total CLA intermediates (mean 4.55, 6.58, 10.9, and 22.0, SEM = 3.94, for 1.0, 2.5, 5.0, and 10.0 mg of added LeA, respectively).
Table 1Adjusted parameters of a single available pool, first-order kinetic model describing the disappearance of linoleic acid (LeA) during incubations with strained ruminal fluid.
Estimated using a first-order exponential model Qt=a+b(1 – e−ct), where Qt is the percentage of LeA disappearance at time t (h), a is the disappearance of LeA at time 0h (% of the initial amount), b is the percentage of LeA that can potentially disappear during incubations with strained ruminal fluid (% of the initial amount), c is the fractional rate of fraction b (expressed in 1/h), and a+b represents the maximum percentage of LeA disappearance (% of the initial amount).
Disappearance during incubations with 10mg of LeA with strained ruminal fluid was linear according the relationship Qt=14.2±0.626 (%)+7.6±0.0102 (%/h) × t (h) (n=6, r2=0.957, P<0.001), where Qt is the percentage of LeA disappearance at time t.
Significance of linear (L) and quadratic (Q) components of the response to LeA addition on first-order kinetic model parameters. Cubic responses to the amount of LeA incubated on model parameters were not significant (P>0.05).
1.0
2.5
5.0
L
Q
a
28.5
23.4
18.6
2.89
0.044
0.710
b
71.2
77.0
87.4
2.07
0.001
0.908
c
0.541
0.553
0.375
0.0349
0.007
0.124
a + b
99.7
100.4
106
1.27
0.006
0.315
1 Estimated using a first-order exponential model Qt = a + b(1 – e−ct), where Qt is the percentage of LeA disappearance at time t (h), a is the disappearance of LeA at time 0 h (% of the initial amount), b is the percentage of LeA that can potentially disappear during incubations with strained ruminal fluid (% of the initial amount), c is the fractional rate of fraction b (expressed in 1/h), and a + b represents the maximum percentage of LeA disappearance (% of the initial amount).
2 Disappearance during incubations with 10 mg of LeA with strained ruminal fluid was linear according the relationship Qt = 14.2 ± 0.626 (%) + 7.6 ± 0.0102 (%/h) × t (h) (n = 6, r2 = 0.957, P < 0.001), where Qt is the percentage of LeA disappearance at time t.
3 Significance of linear (L) and quadratic (Q) components of the response to LeA addition on first-order kinetic model parameters. Cubic responses to the amount of LeA incubated on model parameters were not significant (P > 0.05).
Figure 3Temporal changes in the disappearance of linoleic acid (LA) during incubations of 1.0 (●), 2.5 (○), 5.0 (▴), and 10.0 (▵) mg of added linoleic acid with strained ruminal fluid. Values represent least squares means (SEM = 4.97%).
Table 2Effect of incremental addition of linoleic acid (LeA) on mean accumulation of biohydrogenation intermediates and end products during incubations with strained ruminal fluid.
Values represent least squares means measured as a difference relative to the control determined after 0-, 1.5-, 3.0-, 4.5-, 6.0-, and 9.0-h incubations (n=5). CLA=conjugated linoleic acid.
Significance of linear (L) and quadratic (Q) components of the response to LeA addition. Cubic responses to the amount of LeA incubated were not significant (P>0.05).
Refers to 18:2 intermediates, excluding isomers of CLA and LeA.
78.9
128
322
963
51.5
<0.001
0.020
18:1
291
795
1,855
3,517
149.8
<0.001
0.505
18:0
402
804
1,193
344
187.6
0.289
<0.001
1 Values represent least squares means measured as a difference relative to the control determined after 0-, 1.5-, 3.0-, 4.5-, 6.0-, and 9.0-h incubations (n = 5). CLA = conjugated linoleic acid.
2 Significance of linear (L) and quadratic (Q) components of the response to LeA addition. Cubic responses to the amount of LeA incubated were not significant (P > 0.05).
3 Refers to 18:2 intermediates, excluding isomers of CLA and LeA.
Figure 4Temporal changes in the accumulation of (A) linoleic acid (LeA); (B) conjugated linoleic acid (CLA); (C) nonconjugated 18:2 (NC 18:2); (D) trans 18:1; (E) cis 18:1; and (F) stearic acid (18:0) during incubations of 1.0 (●), 2.5 (○), 5.0 (▴), and 10.0 (▵) mg of LeA with strained ruminal fluid. Values represent least squares means (n = 5; SEM = 0.34, 0.30, 0.06, 0.27, 0.17, and 0.29 mg/flask for LeA, CLA, NC 18:2, trans 18:1, cis 18:1, and 18:0, respectively).
Incremental addition of LeA resulted in a linear (P < 0.01) increase in the accumulation of cis-9,trans-11 CLA and trans-10,cis-12 CLA (Table 3) during incubations with strained ruminal fluid. In addition to these 2 main conjugated intermediates, incubations of LeA also increased linearly (P < 0.05) the accumulation of cis-9,cis-11 CLA; cis-10,cis-12 CLA; trans-9,trans-11 CLA; and trans-10,trans-12 CLA (Table 3). Temporal changes in the accumulation of Δ9,11 and Δ10,12 geometric isomers of CLA over 9-h incubations of incremental amounts of LeA with strained ruminal fluid are shown in Figure 5. Formation of cis-9,trans-11 CLA was instantaneous following the addition of LeA to the fermentation flask, and was rapidly hydrogenated, whereas trans-10,cis-12 CLA tended to accumulate with the highest abundance observed after 1.5 h (Figure 5). At the highest level of LeA addition, cis-9,trans-11 CLA accumulated after 3 h (Figure 5).
Table 3Effect of incremental addition of linoleic acid (LeA) on mean accumulation of isomers of conjugated linoleic acid (CLA) during incubations with strained ruminal fluid.
Values represent least squares means measured as a difference relative to the control determined after 0-, 1.5-, 3.0-, 4.5-, 6.0-, and 9.0-h incubations (n=5).
Significance of linear (L), quadratic (Q), and cubic (C) components of the response to LeA addition.
1.0
2.5
5.0
10.0
L
Q
C
cis-9,cis-11 CLA
0.00
0.00
2.24
3.45
0.158
<0.001
0.021
<0.001
cis-10,cis-12 CLA
0.00
0.00
2.96
4.89
0.215
<0.001
0.062
<0.001
cis-12,cis-14 CLA
0.00
0.00
0.751
1.24
0.072
<0.001
0.181
0.009
cis-9,trans-11 CLA
18.9
20.4
24.3
65.2
7.65
<0.001
0.149
0.816
cis-11,trans-13 CLA
0.00
0.00
1.79
1.45
0.312
0.003
0.028
0.052
cis-12,trans-14 CLA
4.57
5.96
1.53
0.55
0.590
<0.001
0.345
0.004
trans-7,cis-9 CLA
0.71
0.24
0.65
0.19
0.171
0.159
0.700
0.051
trans-8,cis-10 CLA
0.00
0.62
2.12
2.00
0.123
<0.001
<0.001
0.016
trans-10,cis-12 CLA
27.6
57.8
86.9
162
7.29
<0.001
0.909
0.408
trans-11,cis-13 CLA
11.0
1.28
0.33
0.26
2.965
0.042
0.053
0.190
trans-12,cis-14 CLA
3.51
2.68
0.36
0.39
0.571
0.001
0.021
0.306
trans-7,trans-9 CLA
0.00
0.36
1.43
0.84
0.170
0.002
<0.001
0.092
trans-8,trans-10 CLA
2.64
1.67
3.17
2.42
0.497
0.711
0.524
0.036
trans-9,trans-11 CLA
5.26
2.41
6.04
9.22
1.116
0.004
0.276
0.061
trans-10,trans-12 CLA
12.0
10.3
26.4
21.4
3.35
0.021
0.053
0.049
trans-11,trans-13 CLA
42.3
46.7
20.8
29.1
7.49
0.088
0.143
0.100
trans-12,trans-14 CLA
8.48
11.0
1.84
2.02
1.888
0.003
0.207
0.018
trans-13,trans-15 CLA
2.34
1.30
0.24
0.17
0.341
<0.001
0.012
0.915
1 Values represent least squares means measured as a difference relative to the control determined after 0-, 1.5-, 3.0-, 4.5-, 6.0-, and 9.0-h incubations (n = 5).
2 Significance of linear (L), quadratic (Q), and cubic (C) components of the response to LeA addition.
Analysis of FAME, prepared from NEFA extracted from fermentation flasks, revealed the appearance of several unknown peaks eluting between 19:0 and LeA in the GC chromatogram (Figure 6). Initial identification of unusual fatty acids eluting in this region was based on retention time comparisons with authentic standards, but because of the limited number of reference materials, few peaks could be identified with a high degree of confidence. The GC-MS analysis of novel intermediates as methyl esters revealed that all exhibited a molecular ion at m/z 294, confirming an 18-carbon structure with 2 double bonds. Fractionation of FAME by Ag+-TLC enabled 6 distinct bands to be separated, corresponding to methyl esters of saturated fatty acids, trans-monoenoic acids, and cis-monoenoic acids, and mixtures of trans,trans; trans,cis; cis,trans; and cis,cis dienoic acids. Partial GC chromatograms indicating the separation of 18:2 isomers recovered after Ag+-TLC fractionation are shown in Figure 7.
Figure 6Partial gas chromatogram indicating the separation of 18:2 isomers formed during 4.5-h incubations of 10 mg of linoleic acid with strained ruminal fluid. Isomers were identified based on silver-ion thin-layer chromatography fractionation of fatty acid methyl esters and electron impact ionization spectra obtained by GC-MS analysis of corresponding 4,4-dimethyloxaline derivatives. Peak identification: 1 = 19:0; 2 = trans-11,trans-15 18:2; 3 = trans-9,trans-14 18:2; 4 = trans-9,trans-12 18:2; 5 = unresolved trans-8,cis-12 18:2 and ▵4,12 18:2; 6 = cis-5,cis-12 18:2; 7 = cis-6,cis-12 18:2; 8 = cis-7,cis-12 18:2; 9 = cis-9,trans-12 18:2; 10 = cis-8,cis-12 18:2; 11 = trans-9,cis-12 18:2; 12 = trans-11,cis-15 18:2; and 13 = cis-9,cis-12 18:2.
The structure of unusual 18:2 isomers were formally identified based on GC-MS analysis of DMOX derivatives prepared from each FAME fraction recovered after Ag+-TLC. Characteristic ion fragments in the mass spectrum of DMOX derivatives used to identify the structure of unusual 18:2 isomers are listed in Table 4. Spectra of all DMOX derivatives of 18:2 isomers contained abundant ions at m/z 113 and 126 and a molecular ion at m/z 333 (Table 4). A prominent fragment at m/z at 113 arises from a McLafferty rearrangement ion formed by the migration of the γ-hydrogen, followed by cleavage between carbon atoms 2 and 3, whereas the abundant ion at m/z at 126 is thought to be formed due to a cyclization-displacement reaction and cleavage between carbon atoms 4 and 5 (
Structure analysis of fatty acids by gas chromatography-low resolution electron impact mass spectrometry of their 4, 4-dimethyloxazoline derivatives—A review.
). The mass spectrum of the DMOX derivative of cis-7,cis-12 18:2 contained 2 pairs of ion fragments separated by 12-amu gaps at m/z 168 and 180, and 236 and 248, and 14-amu intervals between ions at m/z 194, 208, and 222, and 262, 276, 290, 304, and 318 locating double bonds at Δ7 and Δ12 (Figure 8A). Double-bond positions for isomers of Δ8,12 18:2 were indicated based on 12-amu intervals between m/z 182 and 194, and 236 and 248 (Figure 8B), and a prominent ion at m/z 222 that represents cleavage between carbon atoms 9 and 10 (i.e., at the center of the dimethylene-interrupted ethylenic bond system).
Table 4Characteristic ion fragments recorded during GC-MS analysis of 4,4-dimethyloxazoline derivatives of unusual octadecadienoic acids formed during incubations of linoleic acid with strained ruminal fluid.
Fatty acid
Characteristic ion fragments (m/z, relative intensity)
Mass spectra were recorded at an ionization energy of 70eV during analysis of 4,4-dimethyloxazoline derivatives on a 100-m CP-Sil 88 fused silica capillary column (Chrompack International BV, Middelburg, the Netherlands) using a temperature gradient program and helium as the carrier gas operated at constant pressure (142.6 kPa) at a flow rate of 0.6 mL/min.
1 Mass spectra were recorded at an ionization energy of 70 eV during analysis of 4,4-dimethyloxazoline derivatives on a 100-m CP-Sil 88 fused silica capillary column (Chrompack International BV, Middelburg, the Netherlands) using a temperature gradient program and helium as the carrier gas operated at constant pressure (142.6 kPa) at a flow rate of 0.6 mL/min.
Figure 8Mass spectrum of the 4,4-dimethyloxazoline derivative of (A) cis-7,cis-12 18:2 and (B) cis-8,cis-12 18:2, formed during incubations of linoleic acid with strained ruminal fluid.
Because GC-MS analysis of DMOX derivatives does not discriminate between geometric isomers, the double bond geometry of most NC 18:2 intermediates could be confirmed based on the appearance of FAME in bands separated by Ag+-TLC. However, the methyl ester of Δ4,12 18:2 was not recovered in either cis,cis 18:2 fraction (Figure 7) suggesting this isomer contained at least one trans double bond, whereas the retention time and relative elution order of this isomer during GC analysis of FAME (Figure 6) would tend to implicate a cis,cis double bond configuration.
Complementary Ag+-TLC and GC-MS analysis of DMOX derivatives confirmed that the biohydrogenation of LeA by strained ruminal fluid under the specified conditions of this experiment resulted in the formation of Δ4,12 18:2; cis-5,cis-12 18:2; cis-6,cis-12 18:2; cis-7,cis-12 18:2; cis-8,cis-12 18:2; cis-9,trans-12 18:2; trans-8,cis-12 18:2; and trans-9,cis-12 18:2. Addition of incremental amounts of LeA resulted in linear or quadratic (P < 0.01) increases in the accumulation of cis-5,cis-12 18:2 + cis-6,cis-12 18:2; cis-7,cis-12 18:2; cis-8,cis-12 18:2; trans-8,cis-12 18:2 + Δ4,12 18:2; and trans-9,cis-12 18:2 during incubations with strained ruminal fluid (Table 5). Increases in the amount of these biohydrogenation intermediates during incubations with 1.0, 2.5, and 5.0 mg of LeA reached a plateau after 1.5 or 3 h, but decreased thereafter (Figure 9). A mixture of trans-8,cis-12 18:2 and Δ4,12 was found to accumulate immediately after LeA was added to strained ruminal fluid. Incubations with 10 mg of LeA resulted in the accumulation of all identified NC 18:2 isomers over time (Figure 9).
Table 5Effect of incremental addition of linoleic acid (LeA) on mean accumulation of nonconjugated octadecadienoic acids during incubations with strained ruminal fluid.
Values represent least squares means measured as a difference relative to the control determined after 0-, 1.5-, 3.0-, 4.5-, 6.0-, and 9.0-h incubations (n=5).
Significance of linear (L) and quadratic (Q) components of the response to LeA addition. Cubic responses to the amount of LeA incubated were not significant (P>0.05).
1 Values represent least squares means measured as a difference relative to the control determined after 0-, 1.5-, 3.0-, 4.5-, 6.0-, and 9.0-h incubations (n = 5).
2 Significance of linear (L) and quadratic (Q) components of the response to LeA addition. Cubic responses to the amount of LeA incubated were not significant (P > 0.05).
3 Contains cis-5,cis-12 18:2 as a minor component.
Figure 9Temporal changes in the accumulation of (A) cis-7,cis-12 18:2; (B) cis-8,cis-12 18:2; (C) unresolved trans-8,cis-12 and 4,12 18:2; and (D) trans-9,cis-12 18:2 during incubations of 1.0 (●), 2.5 (○), 5.0 (▴), and 10.0 (Δ) mg of linoleic acid with strained ruminal fluid. Values represent least squares means (n = 5; SEM = 0.033, 0.052, 0.027, and 0.017 mg/flask for cis-7,cis-12 18:2; cis-8,cis-12 18:2; trans-8,cis-12 + 4,12 18:2; and trans-9,cis-12 18:2, respectively).
Isomers of 18:1 represented the most abundant intermediates to accumulate during incubations of LeA with strained ruminal fluid (Table 6). Addition of 1.0, 2.5, and 5.0 mg of LeA resulted in the appearance of cis-9 18:1; cis-12 18:1; trans-10 18:1; trans-11 18:1; and trans-13,14 18:1 as the main 18:1 intermediates, whereas cis-9 18:1, cis-12 18:1, trans-10 18:1, and trans-11 18:1 were the major 18:1 isomers to accumulate during incubations with 10 mg of LeA. Temporal changes in the accumulation of cis-9 18:1, cis-12 18:1, trans-9 18:1, trans-10 18:1, trans-11 18:1, and trans-12 18:1 are shown in Figure 10. Following the addition of LeA to the fermentation flask, cis-9 18:1 was found to be formed instantaneously and subsequently hydrogenated, whereas cis-12 18:1 accumulated after 1.5 h, depending on the amount of added LeA (Figure 10). Addition of 10 mg of LeA caused cis-12 18:1 to accumulate over the course of the 9-h incubations (Figure 10). Incubation of 1.0, 2.5, and 5.0 mg of LeA was associated with an initial increase in the amounts of trans-10 18:1 and trans-11 18:1 in fermentation flasks, but the amounts remained relatively constant after 1.5 h. In contrast, addition of 10.0 mg of LeA resulted in a gradual increase in trans-10 18:1 in fermentation flasks up to 4.5 h and the progressive accumulation of trans-11 18:1 during the course of incubations with strained ruminal fluid. Increasing the amounts of added LeA resulted in a linear (P = 0.021) increase in the mean ratio of trans-10 18:1:trans-11 18:1 in fermentation flasks over the 9-h incubation period (0.787, 0.889, 1.076, and 1.671, SEM = 0.315, for 1.0, 2.5, 5.0, and 10.0 mg of added LeA, respectively). Incubations of LeA with strained rumen fluid also resulted in a dose-dependent increase (P < 0.01) in the accumulation of trans-6, -7, -8, -9, -12, -13 and, -14 18:1 (Table 6). Changes in the accumulation of trans-6, -7, and -8 18:1 and trans-9 18:1 over the course of incubations with LeA followed the same pattern as observed for trans-10 18:1 (data not presented). At lower levels of LeA addition, trans-12 18:1 accumulated in fermentation flasks after 3 h, with the amounts remaining relatively constant thereafter, whereas the addition of 10 mg of LeA resulted in a cumulative increase in trans-12 18:1 over time (Figure 10). Temporal changes in the appearance of trans-13 and -14 18:1 followed the same pattern as trans-12 18:1 (data not presented).
Table 6Effect of incremental addition of linoleic acid (LeA) on mean accumulation of octadecenoic acids during incubations with strained ruminal fluid.
Values represent least squares means measured as a difference relative to the control determined after 0-, 1.5-, 3.0-, 4.5-, 6.0-, and 9.0-h incubations (n=5)
Significance of linear (L) and quadratic (Q) components of the response to LeA addition. Cubic responses to the amount of LeA incubated were not significant (P>0.05).
1.0
2.5
5.0
10.0
L
Q
cis-9 18:1
39.4
124
273
1,070
39.7
<0.001
<0.001
cis-11 18:1
11.5
14.2
23.9
59.3
5.30
<0.001
0.072
cis-12 18:1
78.9
219
488
828
61.2
<0.001
0.353
trans-6, -7, -8 18:1
10.4
28.4
75.9
92.0
12.14
<0.001
0.053
trans-9 18:1
6.42
23.6
59.9
99.2
8.31
<0.001
0.197
trans-10 18:1
23.0
65.9
223
475
24.1
<0.001
0.785
trans-11 18:1
40.8
96.6
220
530
32.5
<0.001
0.321
trans-12 18:1
12.9
41.7
99.3
98.2
17.94
<0.001
0.018
trans-13, -14 18:1
35.3
104
209
176
32.3
0.002
0.003
trans-15 18:1
17.9
43.5
82.3
65.9
10.22
0.002
0.001
trans-16 18:1
17.9
37.7
64.5
27.0
8.56
0.622
<0.001
1 Values represent least squares means measured as a difference relative to the control determined after 0-, 1.5-, 3.0-, 4.5-, 6.0-, and 9.0-h incubations (n = 5)
2 Significance of linear (L) and quadratic (Q) components of the response to LeA addition. Cubic responses to the amount of LeA incubated were not significant (P > 0.05).
Estimation of transfer rates among fatty acid pools based on the appearance of biohydrogenation intermediates during incubations of 1.0 and 2.5 mg of LeA with strained ruminal fluid using the multiple-pool, first-order kinetic model were not possible due to a small CLA pool size, principally due to the lack of cis-9,trans-11 CLA accumulation in fermentation flasks (Figure 5). Despite attempts to estimate the percentage of added LeA initially isomerized to cis-9,trans-11 CLA transfer rates between pools could not be resolved. Accumulation of intermediates formed during incubations of 5 and 10 mg of LeA with strained ruminal fluid were able to be fitted to the dynamic multi-compartmental model (Figure 1), and indicated that the transfer rate of LeA to the CLA pool (mean 19.1 and 7.3%/h, respectively) was almost identical to the rate of LeA transfer to the NC 18:2 isomer pool (corresponding values 18.1 and 7.3%/h). However, the rates of transfer from the CLA to 18:1 pool (138 and 103%/h, for 5.0 and 10.0 mg of LeA, respectively) were higher compared with the corresponding rates of transfer from the NC 18:2 to 18:1 pool (63.7 and 20.2%/h). Furthermore, increases in LeA addition had much smaller inhibitory effects on the rate of transfer from the CLA to 18:1 pool compared with the rate of transfer from the NC 18:2 to 18:1 pool. The transfer rates from the 18:1 to 18:0 pool were estimated to be lower than other steps of LeA biohydrogenation by strained ruminal fluid and found to be decreased by increases in LeA addition (14.6 and 1.50%/h for incubations with 5.0 and 10.0 mg of LeA, respectively).
Discussion
Fatty Acid Biohydrogenation In Vitro
Numerous in vitro and in vivo studies have enabled the major pathways of ruminal LeA biohydrogenation to be elucidated (
). However, no single experiment has provided a comprehensive evaluation of the intermediates that accumulate during incubations of LeA with strained ruminal fluid and the relative rates of their formation and reduction.
In the current experiment, a batch culture method was used to examine the fate of LeA during incubations with strained ruminal fluid over a range of doses that could be expected under physiological conditions. The amounts of added LeA varied between 20 and 200 mg/L, which is in the range of concentrations of LeA in ruminal digesta of grazing animals (
). High concentrations of LeA are known to inhibit microbial growth, depending on bacterial species, and arrest the hydrogenation of unsaturated fatty acids (
). Potential inhibitory effects of LeA on microbial growth were minimized in this study by dispersing LeA in a sufficient concentration of polyoxyethylene sorbitan monooleate detergent (Tween 80) that is known to decrease the toxic effects of LeA on Propionibacterium freudenreichii ssp. shermanii JS (
). A recent comparison also reported that emulsions containing LeA prepared using Tween 80, rather than ethanol or sonication, results in more extensive hydrogenation of LeA, greater accumulation of 18:1 intermediates, and higher 18:0 production during incubations with rumen fluid (
), with the implication that the use of surfactant does not cause substantial interference in the biohydrogenation of LeA in vitro.
Incubations were limited up to 9 h, to avoid potential losses in the activity of microbes in rumen fluid, which can result in the production of biohydrogenation intermediates in vitro being erroneous with respect to ruminal lipid metabolism in vivo (
). Measurements of fatty acid flow at the duodenum combined with rumen evacuations have indicated that LeA is hydrogenated at a rate of between 14.6 and 16.7%/h in the rumen of lactating cows (
), indicating that the duration of in vitro incubations in this experiment extend across the range of ruminal LeA turnover rates estimated in vivo.
Previous studies have demonstrated that the amount of added substrate is the major determinant of the rate and extent of LeA biohydrogenation in vitro, whereas the composition of diet or the amount of lipid supplements fed to donor animals may also influence the disappearance of added LeA during incubations with rumen fluid (
). In the current experiment, rumen fluid was sampled from the dorsal sac at the same time each day from a nonlactating cow fed a diet containing relatively low amounts of lipid, and incubations were completed over a 10-d period to minimize between-day variations in the composition of rumen fluid. Because the adsorption onto feed particles is considered the major site of dietary unsaturated fatty acid hydrogenation by bacteria in the rumen (
), LeA was added directly on top of the ground hay before the addition of rumen fluid to mimic conditions in vivo.
Formation of CLA
Incubations of incremental amounts of LeA with strained rumen fluid over periods from 0 to 9 h confirmed that one of the pathways of LeA biohydrogenation involved the formation of cis-9,trans-11 CLA and subsequent reduction to trans-11 18:1 and 18:0, consistent with the findings of much earlier studies (
). However, several geometric isomers of Δ9,11 and Δ10,12 CLA were found to accumulate, with trans-10,cis-12 CLA being the other major conjugated intermediate, consistent with previous investigations of LeA biohydrogenation by mixed ruminal microbiota or pure strains of ruminal bacteria (
Characterization of 18:1 and 18:2 isomers produced during microbial biohydrogenation of unsaturated fatty acids from canola and soya bean oil in the rumen of lactating cows.
J. Anim. Physiol. Anim. Nutr. (Berl.).2002; 86: 422-432
Classical studies of fatty acid metabolism by rumen bacteria have considered that the first committed step of LeA hydrogenation proceeds via the isomerization of the cis-12 double bond (
). More recent studies have provided evidence that hydrogenation of LeA by rumen bacteria occurs via 2 distinct mechanisms, one involving direct isomerization, yielding geometric isomers of Δ10,12 CLA, the other resulting in the formation of geometric Δ9,11 CLA isomers, possibly mediated by a hydrogen-abstraction catalyzed reaction (
). The same mechanisms also appear to account for the formation of Δ9,11 CLA and Δ10,12 CLA isomers during incubations of LeA with bacteria isolated from the human intestine (
). An additional mechanism for the conversion of LeA to cis-9,trans-11 CLA by certain human gut-colonizing bacteria has been suggested to involve sequential hydration and dehydration reactions via the formation of 10-OH, cis-12 18:1 (
). In this experiment, formation of cis-9,trans-11 CLA was instantaneous following the addition of LeA to the fermentation flask and was rapidly hydrogenated, whereas trans-10,cis-12 CLA tended to accumulate, reaching a plateau after 1.5 h of incubation. Appearance of cis-9, trans-11 CLA during incubations of LeA with rumen fluid is known to be transient and much lower relative to other intermediates and end products (
Characterization of 18:1 and 18:2 isomers produced during microbial biohydrogenation of unsaturated fatty acids from canola and soya bean oil in the rumen of lactating cows.
J. Anim. Physiol. Anim. Nutr. (Berl.).2002; 86: 422-432
). Under certain conditions, cis-9,trans-11 CLA and trans-10,cis-12 CLA have been reported to accumulate during incubations of LeA with mixed rumen microbes (
). Differences with respect to the accumulation of CLA isomers in this and earlier studies appear to be related to both the methods used to introduce LeA into incubation vessels and the amount of added substrate. Flask contents were devoid of trans-9,cis-11 CLA during incubations with LeA in this experiment. Earlier reports have shown that the addition of LA (50–500 mg/L) result in minor amounts of trans-9, cis-11 CLA being formed during incubations with mixed ovine ruminal microbes, Butyrivibrio fibrisolvens and Clostridium proteoclasticum P-18 (
Fermentation flasks were also found to contain small amounts of trans-7,cis-9 CLA that were independent of the amount of added LeA. In contrast, analysis of lipid extracted from ruminal fluid (
Duodenal and milk trans octadecenoic acid and conjugated linoleic acid (CLA) isomers indicate that postabsorptive synthesis is the predominant source of cis-9-containing CLA in lactating dairy cows.
) digesta in cattle has been shown to be devoid of trans-7,cis-9 CLA. The reasons for the differences between in vitro and in vivo reports are not obvious, but there was no indication from this investigation that trans-7,cis-9 CLA is an intermediate of LeA biohydrogenation.
Formation of Nonconjugated Octadecadienoic Intermediates
Incubations of LeA with ruminal fluid have been reported to result in the accumulation of cis-9,trans-12 18:2; trans-9,cis-12 18:2; and trans-9,trans-12 18:2 isomers (
Characterization of 18:1 and 18:2 isomers produced during microbial biohydrogenation of unsaturated fatty acids from canola and soya bean oil in the rumen of lactating cows.
J. Anim. Physiol. Anim. Nutr. (Berl.).2002; 86: 422-432
). During incubations of LeA with strained ruminal fluid in this experiment, several unusual 18:2 intermediates, identified as ▵4,12 18:2; cis-5,cis-12 18:2; cis-6,cis-12 18:2; cis-7,cis-12 18:2; cis-8,cis-12 18:2; and trans-8,cis-12 18:2, were also found to accumulate.
Prior to the analysis of flask contents, the methods used for lipid extraction and methylation of NEFA were evaluated extensively using authentic FAME and NEFA standards. Based on these investigations, no indications were found that these procedures caused isomerization, hydration, or oxidization of unsaturated fatty acids, including LeA. Therefore, the appearance and accumulation of unusual cis, cis octadecadienoic acids, at least under the specified conditions of this experiment, occur during the hydrogenation of LeA. No reports have been made of ▵4,12 18:2; cis-5,cis-12 18:2; cis-6,cis-12 18:2; cis-7,cis-12 18:2; or cis-8,cis-12 18:2 concentrations in ruminal digesta, but cis-6,cis-10 18:2 has been detected in butter oil (
The appearance of minor 18:2 intermediates containing a cis-12 double bond also provides evidence that biohydrogenation of LeA also proceeds by a mechanism other than hydrogen abstraction or isomerization of the cis-12 double bond (
). Several NC 18:2 isomers, including cis-8,cis-12 18:2; cis-8,trans-12 18:2; cis-9,cis-13 18:2; and cis-9,trans-13 18:2, are formed during hydrogenation of LeA with iridium, palladium, and ruthenium catalysts as a result of the release of the hydrogen atom from the adjacent methylene group at Δ8 or Δ 14 of the semi-hydrogenated C–C bond and rotation of the C–C bond during abstraction of the hydrogen atom (
). Further investigations are required to elucidate mechanisms responsible for the formation of minor intermediates during the hydrogenation of LeA with strained ruminal fluid.
Formation of Octadecenoic Intermediates
Incremental addition of LeA resulted in the accumulation of several 18:1 intermediates in fermentation flasks with cis-9 18:1, cis-12 18:1, trans-10 18:1, and trans-11 18:1 as the main products, consistent with previous observations (
). The appearance of cis-9 18:1 and cis-12 18:1 formed during incubations with ruminal fluid could be considered as evidence of LeA biohydrogenation involving the direct reduction of cis-9 and cis-12 double bonds, but it is also possible that these 18:1 intermediates originate from the hydrogenation of 18:2 intermediates. During incubations of LeA with strained ruminal fluid, trans-10,cis-12 CLA was found to accumulate before that of cis-12 18:1, trans-10 18:1, and trans-12 18:1. These findings are consistent with reports that pure cultures of ruminal Butyrivibrio fibrisolvens hydrogenate a mixture of trans-10,cis-12 CLA and trans-10,trans-12 CLA to cis-12 18:1, trans-10 18:1, and trans-12 18:1 (
), and ruminal infusions of a mixture of CLA isomers containing trans-10,cis-12 CLA result in a dose-dependent increase in plasma and milk fat trans-10 18:1 concentrations in lactating cows (
Alterations in blood plasma and milk fatty acid profiles of lactating Holstein cows in response to ruminal infusion of a conjugated linoleic acid mixture.
Both trans-10 18:1 and trans-11 18:1 were found to accumulate, but the addition of greater amounts of LeA resulted in a linear increase in the ratio of trans-10 18:1 to trans-11 18:1. In earlier studies, the ratio of trans-10 18:1 to trans-11 18:1 has been shown to increase 2-fold over 12 h during incubations of LeA with rumen bacteria but remain unchanged with rumen protozoa or mixed microbes (
). Changes in the formation and accumulation of these trans 18:1 intermediates have been reported in cows fed high-concentrate diets or low-forage diets containing oils rich in polyunsaturated fatty acids (
Duodenal and milk trans octadecenoic acid and conjugated linoleic acid (CLA) isomers indicate that postabsorptive synthesis is the predominant source of cis-9-containing CLA in lactating dairy cows.
Biohydrogenation, duodenal flow, and intestinal digestibility of trans fatty acids and conjugated linoleic acids in response to dietary forage concentrate ratio and linseed oil in dairy cows.
Under the controlled conditions of this experiment, the shift toward trans-10 at the expense of trans-11 18:1 occurred over a relatively short incubation period. Previous reports have indicated that decreases in pH promote the formation of trans-10,cis-12 CLA and trans-10 18:1 from LeA by ruminal microbes in vitro (
). However, the mean decrease in pH over the course of incubations in this experiment was marginal (0.4 unit) and independent of the amount of LeA added. Therefore, the changes in the ratio of trans-10 18:1 to trans-11 18:1 in the present investigation are not readily explained by differences in rates of Δ9,11 CLA and Δ10,12 CLA formation but may reflect time-dependent changes in the relative rates in the reduction of 18:2 intermediates. Recent studies examining the products formed during hydrogenation of CLA isomers by Butyrivibrio fibrisolvens suggest that the reduction of geometric isomers of Δ9,11 CLA occurs via a different mechanism responsible for the hydrogenation of other 18-carbon unsaturated fatty acids (
Incubations with strains of Enterococcus fecalis isolated from the rumen indicated that LeA may also be hydrated to yield 10-OH, cis-12 18:1, and cis-9, 13-OH 18:1 (
), whereas the reduction of cis-9 18:1 and trans-11 18:1 by Propionibacterium acnes involves the formation of 10-O-18:0 via an 10-OH-18:0 intermediate (
). Analysis of FAME by GC did not permit the quantification of oxygenated 18-carbon fatty acids in this experiment. However, the amount of 18-carbon fatty acids in fermentation flasks (1.15, 2.50, 4.97, and 10.08 mg/flask) following the addition of 1.0, 2.5, 5.0, and 10.0 mg of LeA, respectively, indicates that hydration did not represent a major route of LeA transformation in this study.
Reduction of Octadecenoic Intermediates to Stearic Acid
Conversion of trans 18:1 to 18:0 is known to be inhibited by high LeA concentrations, an effect attributed to irreversible substrate (
Biohydrogenation of unsaturated fatty acids III. Purification and properties of a linoleate Δ12-cis,Δ11-trans-isomerase from Butyrivibrio fibrisolvens.
) of octadecenoic acid reductase activity, or a combination of both mechanisms. Recent studies have shown that the growth of Clostridium proteoclasticum P-18, a ruminal bacterium capable of producing 18:0, can be inhibited during incubations with trans 18:1 and demonstrated that the more unsaturated fatty acids exert more toxic effects on the ruminal bacteria involved in biohydrogenation (
). However, the rates of disappearance and appearance of CLA isomers during incubations with 10 mg of LeA in the present study were constant, indicating that neither a decrease in substrate concentrations nor the accumulation of trans and cis intermediates altered the initial rate of LeA biohydrogenation with the implication that the rate of the initial isomerization of LeA is finite, consistent with previous considerations (
). Furthermore, incubations of 5 and 10 mg of LeA with strained ruminal fluid caused NC 18:2 and 18:1 intermediates to accumulate over time, which could be interpreted as evidence that NC 18:2 isomers inhibit the reduction of 18:1 to 18:0.
) was used to estimate the rates of transfer from various fatty acid pools to provide further insight into the kinetics of LeA biohydrogenation. Modeling of the appearance of intermediates during incubations with 5.0 and 10.0 mg of LeA indicated that the rates of transfer of LeA to the CLA pool were almost identical to the transfer from the LeA to NC 18:2 pool, suggesting that these reactions, which involve different mechanisms, may occur at the same rate. However, the CLA pool was, in the main, comprised of trans-10,cis-12 CLA, whereas the conversion of LeA to cis-9,trans-11 CLA was found to occur almost instantaneously. It would, therefore, appear that the kinetics of LeA isomerization to 10,12 geometric isomers of CLA are similar to the rates of LeA conversion to NC 18:2.
Transfer rates from the CLA pool to 18:1 pool were higher compared with the rate of transfer from the NC 18:2 to 18:1 pool. However, estimates of transfer of LeA to CLA and from CLA to 18:1 pools have to be interpreted with caution, and considered more apparent than true, because of the limited accumulation of CLA isomers over time and instantaneous formation of Δ9,11 CLA and Δ10,12 CLA immediately after the addition of LeA to incubation flasks. As a result, the rate of CLA isomer reduction in this experiment was 7.3 and 19.1%/h, being lower than values of 23.5 to 27.4%/h, when the production of CLA did not account for LeA being converted to NC 18:2 intermediates (
). Transfer of LeA to the NC 18:2 isomer pool was estimated to vary between 7.3 and 18.1%/h, depending on the amount of added LeA, which would account for the differences in the initial rates of LeA isomerization between the present and earlier experiments.
Estimates of transfer from the 18:1 pool to 18:0 were much lower compared with fractional rates for other steps of LeA biohydrogenation and decreased in direct relation to the amount of LeA incubated, in agreement with earlier reports (
). Overall, the use of a dynamic model as well as considerations of temporal changes in intermediates formed during incubations of LeA with strained ruminal fluid indicated that the final reduction of 18:1 intermediates as rate limiting, and suggest that the reduction of CLA isomers occurs at a faster rate than the reduction of NC 18:2 intermediates.
Conclusions
Disappearance of LeA during incubations with strained rumen fluid was dependent on the amount of the added substrate. Current data indicated that LeA biohydrogenation may proceed via the formation of cis-9,trans-11 CLA and serial reduction to trans-11 18:1 and 18:0, but other metabolic pathways are also involved. Biohydrogenation of LeA also resulted in the formation of other geometric isomers of Δ9,11 CLA and Δ10,12 CLA, and caused cis and trans isomers of Δ9, 10, 11, and 12 18:1 to accumulate. Several intermediates, including cis-5,cis-12 18:2; cis-7,cis-12 18:2; and cis-8,cis-12 18:2 were also formed, providing evidence that the biohydrogenation of LeA may also proceed via mechanisms other than hydrogen abstraction or isomerization of the cis-12 double bond. Modeling the kinetics of LeA biohydrogenation indicated that reduction of 18:1 intermediates is rate limiting, and that the reduction of CLA isomers to 18:1 intermediates occurs at a faster rate than the reduction of NC 18:2 intermediates.
Acknowledgements
The authors gratefully acknowledge and appreciate the assistance of Minna Aalto, Piia Kairenius, and Laura Ventto (MTT Agrifood Research Finland) during lipid analysis.
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