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Department of Animal and Veterinary Sciences, Clemson University, Clemson, SC 29634
3 To whom correspondence should be addressed. E-mail: tjnkns{at}Clemson.edu.
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ABSTRACT |
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 TOP ABSTRACT METHODS AND MATERIALSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • hydroxystearic acid • ketostearic acid • rumen • oleic acid
Unsaturated fatty acids comprise a high percentage of the total fatty acids in plant material consumed by ruminant species. The microbial population that inhabits the rumen transforms dietary unsaturated fatty acids into an array of trans fatty acids, conjugated acids, and stearic acid. However, many of the published pathways of microbial fatty acid transformation do not account for the full range of intermediates known to accumulate in ruminal contents.
Oleic acid is a major unsaturated fatty acid in ruminant feeds, but the nature of its transformation within ruminal contents is uncertain. By some accounts, oleic acid transformation by ruminal microorganisms yields only stearic acid without the formation of any intermediates (1,2). Other studies reported the conversion of oleic acid to a variety of trans monenes (3) and to hydroxystearic acid (HSA)5 and ketostearic acid (KSA) by ruminal microorganisms (4–6).
Although formation of HSA and KSA has been documented by some species of ruminal microorganisms, little is known about the quantitative significance or variability of their production in ruminal contents. Documenting their production within ruminal contents would have several important implications. First, failure to account for their synthesis would explain some of the net loss of C18 fatty acids sometimes reported in the rumen, despite previous reports that C18 fatty acids are not absorbed across the ruminal epithelium or catabolized as energy sources by ruminal anaerobes (7). A second implication is that their production within ruminal contents may lead to the entry of HSA and KSA into the human food supply via consumption of meat and milk. Hydroxystearic acid was investigated as a potent inhibitor of cell proliferation having antitumor activity (8).
This study was designed to determine the daily production of HSA and KSA in cultures of ruminal contents as sources of unsaturated fatty acid input varied. The key objectives were to determine the major unsaturated fatty acids that were transformed to HSA and KSA by ruminal microorganisms, and to establish their production over 24 h by ruminal microorganisms grown in continuous cultures under environmental conditions that mimicked in vivo rumen conditions.
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METHODS AND MATERIALS |
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TOPABSTRACTMETHODS AND MATERIALSRESULTSDISCUSSIONLITERATURE CITED |
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Samples of mixed microbial population were taken from the rumen of a fistulated Holstein cow and strained through 2 layers of cheesecloth to remove large feed particles. Surgical procedures and the care of ruminally fistulated cows were approved by the Clemson University Animal Care and Use Committee. Fistulated cows were fed a standard mixed diet consisting [dry matter (DM) basis] of 50% forage (corn silage and alfalfa hay, 9:1) and 50% concentrate (corn, soybean meal, minerals, and vitamins). Concentrates contained adequate oleic (cis-9 18:1) or elaidic (trans-9 18:1) acid to supply triplicate fermentation flasks with 0, 71, 106, or 141 µmol of fatty acid. Separate cultures were assayed in triplicate to examine the loss and gain of C18 fatty acids over 24 h after the addition of 56.8 µmol 12-HSA. The strained ruminal fluid was stirred continuously under CO2 and transferred to cultures that were maintained and sampled as described by AbuGhazaleh and Jenkins (9).
Continuous cultures
Two continuous culture experiments were done using fermentation vessels (average 950 mL working volume) constructed according to the basic design described by Teather and Sauer (10). The first continuous culture study, referred to as the isotope experiment, consisted of reanalysis of samples taken from a previously published study (11) in which 1-13C -oleic acid was added to fermenters to examine the distribution of label in stearic acid and various trans-C18:1 positional isomers. Enrichment was also observed in 2 large unidentified peaks, which were later identified by GC-MS in this study as 10-ketostearic acid (10-KSA) and 10-hydroxystearic acid (10-HSA). The second continuous culture experiment, or production experiment, was designed to compare soybean oil and canola oil addition to the fermenters on formation of 10-HSA and 10-KSA.
Isotope experiment. Details of fermenter specifications and isotope dosing were described previously (11). Briefly, 4 dual-flow continuous culture vessels were used in a 4 x 4 Latin square with 10-d periods to evaluate 2 pH (5.5 and 6.5) and 2 liquid dilution rates (0.05 and 0.10 h–1) arranged factorially. Data were averaged across treatments in this study to determine the overall transfer of oleic acid carbons to HSA and KSA across all conditions. A total of 22 g of feed (55% alfalfa pellets and 45% concentrate, DM basis) was introduced daily into the fermenters in 2 equal amounts at 0830 and 1630 h. Diets contained 29 g/kg added oleic acid with an additional 885 µmol of oleic acid in 5 mL of ethanol injected directly into each fermenter on d 1–9 just after the morning feeding. On d 10, 885 µmol of 1-13C-oleic acid replaced the unlabeled oleic acid in the 5 mL injection. Samples of mixed culture contents were taken at 0 h (just before injection of the stable isotope) and again at 24 h after injection of the isotope. Samples were immediately stored at –5°C.
Production experiment. Fermentors were modified slightly from the design described by Teather and Sauer (10) to include a sidearm that angled downward rather than straight out to allow for greater gravitational pull on overflow contents and minimize clogging at higher feeding rates. As a result of this modification, feed was introduced into fermenters at 60 g/d without evidence of clogging. Diets consisted of 500 g/kg alfalfa pellets and 500 g/kg concentrate and contained either 1) no added lipid (control), 2) 50 g/kg soybean oil (SBO), or 3) 50 g/kg canola oil (CO). Ingredients in the control concentrate (g/kg) were: 375 ground corn, 100 soybean meal, 12.2 CaHPO4, 5.6 salt, and 7.4 NaHCO3. Ingredients and concentrations in the SBO and CO diets were the same except for 50 g/kg fat, 314 g/kg ground corn, and 111 g/kg soybean meal to keep the diets isonitrogenous.
Fermentation vessels were inoculated with a mixed microbial population collected from the rumen of a cannulated Holstein cow fed a mixed diet of forage and concentrate (50:50, DM basis). Ruminal contents were filtered through 2 layers of cheesecloth and transferred to the laboratory in a sealed container to maintain anaerobic conditions. Filtered ruminal fluid was then transferred to 3 fermenters set up in a 3 x 3 Latin-square design with 10-d periods. Samples were taken on the last 3 d of each period. Fermenters were purged with CO2 at a mean rate of 20 mL/min throughout the duration of the experiment. Artificial saliva was prepared as 2 separate solutions, which were then mixed proportionally on a daily basis as described by Slyter et al. (12). If needed, the buffer was titrated with 3 mol/L NaOH to maintain the culture pH between 6.0 and 6.5, which was delivered to each fermenter via a peristaltic pump at a fractional liquid dilution rate of 0.10 h–1. The temperature of the fermenters was maintained at 40°C via a circulating water bath. Culture contents were agitated constantly at 
60 rpm, unless otherwise noted.
Fatty acid analysis and GC/MS
Fermenter samples were freeze-dried and converted to methyl esters in sodium methoxide:methanolic HCl as described by Kramer et al. (13). Enrichments of 10-HSA and 10-KSA were determined on a GC equipped with a Saturn II ion trap MS (Varian Instruments) as previously described (11).
Analysis of fermenter outflow fatty acids in the isotope and production experiments was done on a HP5890A GC (Agilent Technologies) equipped with a flame ionization detector and a 30 m x 0.25 mm (0.2-µm film) Supelco 2380 fused silica capillary column. The injector and detector temperatures were held at 250 and 260°C, respectively. The carrier gas was He (20 cm/s) with an inlet pressure of 104 kPa. The column temperature was programmed for 140°C for 3 min, then increased to 220°C at 2°C/min, and held at 220°C for 2 min. Peaks were quantified by comparison with an internal standard (17:0).
Statistical analysis and calculations
Data were analyzed in all batch and continuous culture experiments by ANOVA using the GLM (general linear model) procedure of SAS (14). Means ± pooled SEM taken from ANOVA are shown in the text. Abundance values from the isotope experiment at 0 h (predosing) and 24 h after dosing with 1-13C-oleic acid were analyzed as a completely randomized design. Enrichments were determined as the difference between abundance values post- and predosing of the 13C isotope, which corrected for natural occurrence. Transfer of 13C from oleic acid to HSA or KSA was considered significant if enrichments differed from zero at P < 0.05 by Student's t test. The percentage of 10-HSA and 10-KSA originating from oleic acid transformation was calculated by dividing enrichment for each fatty acid derivative by enrichment for oleic acid and then multiplying by 100.
Fatty acid results from the batch in vitro cultures were corrected to exclude substrate and inoculum fatty acids by subtracting fatty acids in control cultures (with no added lipid) from fatty acids in cultures with added oleic, elaidic, or 12-HSA. Corrected fatty acid results were analyzed as a completely randomized design with mean separation by a least significant difference test when appropriate. Net losses or gains of C18 fatty acids including HSA or KSA were considered significant if values differed from zero at P < 0.05 by Student's t test.
The model for the Latin-square design used in the production experiment included treatment, period, and fermenter effects. Differences among treatment means were determined by least significant difference and were considered significant at P < 0.05. The sample correlation coefficients (r) and their probabilities of significance are shown for linear regressions relating HSA + KSA production to fatty acid input or net loss. Slopes relating the sum of 10-HSA + 10-KSA production with total 18:1 input and oleic acid net loss per day are shown with coefficients of multiple determination.
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RESULTS |
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TOPABSTRACTMETHODS AND MATERIALSRESULTSDISCUSSIONLITERATURE CITED |
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38 min that were previously reported as unidentified (11). Across all culture samples taken on d 10, the first peak was 93.4 ± 9.5 and the second peak was 98.3 ± 4.8 g/kg of total fatty acids (data not shown). MS analysis revealed the identities of the 1st and 2nd peaks as 10-HSA and 10-KSA, respectively. Although pure standards of 10-HSA and 10-KSA could not be located, injection of a 12-HSA methyl ester standard coeluted with the 2nd large peak. Fragmentation patterns by GC-MS verified that the hydroxyl group was located at the C10 position. Large fragment ions characteristic of the hydroxyl group at the C10 position were seen at m/z 201, 169, and 143. Similarly, large fragments at m/z 199, 171, and 141 in the 1st peak corresponded to placement of the keto group at the C10 position. Abundance of the 13C isotope in total culture oleic acid increased (P < 0.05) after dosing with 1-13C-oleic acid (Table 1). 13C abundances also increased (P < 0.05) in both 10-HSA and 10-KSA after dosing. Therefore, enrichments of the hydroxylated and oxygenated acids were both significant (P < 0.05). Based on enrichment comparisons for the stearic acid derivatives compared with oleic acid, it was estimated that most (>80%) of the 10-HSA and 10-KSA present in the 24-h samples originated from oleic acid.
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The output of 10-HSA + 10-KSA from the fermenters was more related to oleic acid input (r = 0.948, P < 0.01) than to linoleic acid input (r = 0.119, P = 0.71), and also better related to oleic acid net loss (r = 0.848, P < 0.01) than to linoleic acid net loss (r = 0.150, P = 0.64). Similar correlations were not done with linolenic acid because of its colinearity with oleic acid. Slopes relating the sum of 10-HSA + 10-KSA production with total C18:1 input and oleic acid net loss per day were 0.14 (R2 = 0.90) and 0.32 (R2 = 0.72), respectively.
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DISCUSSION |
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TOPABSTRACTMETHODS AND MATERIALSRESULTSDISCUSSIONLITERATURE CITED |
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13C enrichment of 10-HSA and 10-KSA in samples from the isotope study showed that both HSA and KSA originated primarily (>85%) from oleic acid. Additional support that oleic acid is the primary carbon source for formation of the derivatives can be seen from the high correlations between production of HSA or KSA and the input of oleic acid into the cultures. When oleic acid was added to the batch in vitro flasks at 71, 106, and 141 µmol, there were linear increases in the production of both 10-HSA and 10-KSA. Unexpectedly, the correlation was less when production of the 2 derivatives was related to oleic acid net loss. Oleic acid net loss stabilized at 48 µmol/d when oleic acid inputs were 106 and 141 µmol/d, but production of 10-HSA and 10-KSA continued to increase. In the production experiment, the outflow of HSA + KSA from the fermenters was closely related to the input and net loss of oleic acid across fat sources, but there was no relation with linoleic acid input or net loss. Mean daily production of HSA + KSA in continuous culture was 14.4 µmol/100 µmol oleic acid input into the cultures or 31.8 µmol/100 µmol oleic acid net loss. Thus, both stearic acid derivatives were appreciable end products of oleic acid transformation by ruminal microorganisms in this investigation.
The conversion of oleic acid into 10-HSA and 10-KSA indicates that ruminal bacteria are not only able to hydrogenate double bonds in the acyl chain, but also to hydrate them. Some aerobic and nonruminal anaerobes were reported to produce HSA and KSA from oleic acid. Under aerobic conditions, Wallen et al. (15) reported the production of 10-HSA from oleic acid by Pseudomonas sp. This was claimed to be the first report of such a conversion and was attributed to hydration of the double bond as the first step in the use of this compound as an energy source. Subsequent work with Pseudomonas strain NRRL 3266 showed that oleic acid was converted to a mixture of 10-HSA and 10-KSA under aerobic conditions, whereas only 10-HSA was produced under anaerobic conditions (16).
Hudson et al. (4) isolated 2 species of ruminal bacteria (Selenomonas ruminantium and Enterococcus faecalis) that were capable of converting oleic acid into 10-HSA. Of 5 bacterial isolates from the sheep rumen, only 1 bacterium (Fusocillus babrahamensis, strain P2/2) was able to convert oleic acid into HSA (17). Under aerobic conditions, Takatori et al. (18) showed that the relative conversion of oleic acid to 10-HSA and 10-KSA by different Staphylococcus species was 61.3 and 23.2%, respectively. Lanser (19) also reported a higher conversion of oleic acid to 10-KSA (90%) under different aerobic conditions.
Although production of HSA and KSA was directly related to oleic acid input in batch cultures, neither derivative was produced after the addition of elaidic acid to cultures. Because elaidic acid addition also increased stearic acid production, but still did not lead to HSA and KSA formation, stearic acid also was eliminated as a parent compound. Trans-18:1 isomers having double bond positions other than C9 were not investigated as possible parent compounds for HSA formation. Mortimer and Niehaus (20) reported conversion of trans-10 18:1 to 10-HSA by a soluble enzyme preparation from a pseudomonad. Limited availability of a pure trans-10 18:1 source prevented examination of a possible trans-10 18:1 to 10-HSA conversion by ruminal microorganisms in this study. Future experiments will be required to assess the extent of this conversion.
Additional in vitro cultures were assayed in this study to examine the fate of HSA once it was formed from oleic acid. The possibilities were that the HSA was converted to other fatty acids, possibly stearic acid, or that no further metabolism of HSA occurred and it simply accumulated in the culture contents. A source of 10-HSA could not be located; thus, the disappearance of 12-HSA over time from ruminal cultures was examined. The results showed that 24% of the initial 57-µmol dose of 12-HSA was lost over 24 h of incubation, with KSA as the only end product identified. Previous work by Kemp et al. (21) reported that HSA produced by a ruminal Fusocillus strain was not metabolized further and accumulated in cultures. Pseudomonads were reported to convert oleic acid to 10-HSA and 10-KSA under aerobic conditions, but only to 10-HSA when grown anaerobically (16). A Flavobacterium species grown aerobically was reported (22) to hydrate oleic acid to 10-HSA with subsequent conversion of the 10-HSA to 10-KSA by a secondary alcohol dehydrogenase. Therefore, compared with several nonruminal microorganisms, the high 10-KSA:10-HSA ratio that occurred in the anaerobic cultures of this study seems unusual. However, the formation and accumulation of KSA from oxidation of HSA in ruminal in vitro cultures was also reported by Katz and Keeney (5).
Using the results of this investigation combined with results from 2 previous studies (3,23) in our laboratory, a working hypothesis (Fig. 1) for oleic acid transformation by ruminal microorganisms was developed. Three main routes of carbon flow are depicted; biohydrogenation, isomerization, and hydration. The study by Mosley et al. (3) showed the appearance of 13C label in a multitude of trans-18:1 positional isomers when cultures of ruminal microorganisms were dosed with 1-13C-oleic acid, thus establishing cis-to-trans isomerization. Label also was transferred to stearic acid, but it was unknown to what extent the transfer occurred directly from the labeled oleic acid vs. through a trans-18:1 intermediate.
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This study showed the conversion of oleic acid to 10-HSA in whole ruminal contents, in agreement with similar conversions reported in Pseudomonas sp. (15) and select species of isolated ruminal bacteria (4,24). The conversion of oleic acid to 10-HSA in pseudomonads was attributed to hydration with the stereospecific addition of the elements of water across the double bond (25). The high degree of enrichment in 10-HSA and 10-KSA after 1-13C-oleic acid dosing and the failure of a trans-9 18:1 supplement to stimulate production of the derivatives showed that HSA and KSA originated from oleic acid transformation by ruminal microorganisms. Further in vitro work in this investigation showed that HSA and KSA were final products of oleic acid transformation and not intermediates eventually converted to other fatty acids.
The results of this study clearly indicate the formation of 10-HSA and 10-KSA from oleic acid transformation by ruminal microorganisms, and show that their accumulation in ruminal contents is directly related to the extent of oleic acid input and transformation in the rumen. Thus, deposition of these stearic acid derivatives in body tissues of the host ruminant animal would be expected, with subsequent entry into the human food supply via meat and milk consumption. Work is currently underway to determine the concentrations of 10-KSA and 10-HSA in the meat and milk supply across a number of ruminant species fed a variety of fat sources. The biological and physiological consequences of HSA and KSA consumption by humans are currently being examined. Some reported effects include HSA as an inhibitor of cell proliferation and viability (26,27), potent cytotoxic effects of HSA on human melanoma development (8), and their role as important metabolites of lipid peroxidation (28).
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FOOTNOTES |
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2 Approved as technical contribution number 5136 of the South Carolina Agricultural Experiment Station, Clemson University. This project was supported by National Research Initiative Competitive Grant No. 2003-35206-12835 from the U.S. Department of Agriculture Cooperative State Research, Education, and Extension Service.
4 Present address: Southern Illinois University Carbondale, Agriculture Bldg Room 119, Carbondale, IL 62901-4417.
5 Abbreviations used: CO, canola oil; DM, dry matter; HSA, hydroxystearic acid; KSA, ketostearic acid; SBO, soybean oil.
Manuscript received 14 October 2005. Initial review completed 15 November 2005. Revision accepted 6 January 2006.
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LITERATURE CITED |
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28. Bertucci C, Hadaib M, Boga C, Calonghi N, Cappadone C, Masotti L. Gas chromatography/mass spectrometric assay of endogenous cellular lipid peroxidation products: quantitative analysis of 9- and 10- hydroxystearic acids. Rapid Commun Mass Spectrom. 2002;16:859–64.[Medline]
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