Evidence for the Initial Steps of DHA Biohydrogenation by Mixed

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Evidence for the initial steps of DHA biohydrogenation by mixed ruminal microorganisms from sheep involves formation of conjugated fatty acids Noelia Aldai, Pierluigi Delmonte, Susana Alves, Rui J.B. Bessa, and John Kramer J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04563 • Publication Date (Web): 01 Jan 2018 Downloaded from http://pubs.acs.org on January 1, 2018

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Journal of Agricultural and Food Chemistry

Evidence for the initial steps of DHA biohydrogenation by mixed ruminal microorganisms from sheep involves formation of conjugated fatty acids

Noelia Aldai1*, Pierluigi Delmonte2, Susana P. Alves3, Rui J. B. Bessa3 and John K.G. Kramer4

1

Department of Pharmacy and Food Sciences, University of the Basque Country (UPV/EHU),

Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spain; 2Office of Regulatory Science, Centre for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, MD, USA; 3

CIISA, Faculty of Veterinary Medicine, University of Lisbon, Av. da Universidade Técnica, 1300-

477 Lisbon, Portugal; 4Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph N1G 5C9, Ontario, Canada (retired).

*Corresponding author (Tel: (+34) 945 014501; Fax: (+34) 945 013014; E-mail: [email protected], [email protected])

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ABSTRACT

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Incubation of DHA with sheep rumen fluid resulted in 80% disappearance in 6h. The products were

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analyzed as their fatty acid (FA) methyl esters by GC-FID on SP-2560 and SLB-IL111 columns.

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The GC-online reduction×GC and GC-MS techniques demonstrated that all DHA metabolites

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retained the C22 structure (no evidence of chain-shortening). Two new transient DHA products

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were identified; mono-trans methylene interrupted-DHA and mono-conjugated DHA (MC-DHA)

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isomers. Identification of MC-DHA was confirmed by their predicted elution using equivalent chain

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length differences from C18 FA, their molecular ions, and the 22:5 products formed which were the

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most abundant at 6h. The 22:5 structures were established by fragmentation of their 4,4-

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dimethyloxazoline derivatives, and all 22:5 products contained an isolated double bond, suggesting

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formation via MC-DHA. The most abundant c4,c7,c10,t14,c19-22:5 appeared to be formed by

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unknown isomerases. Results suggest that the initial biohydrogenation of DHA was analogous to

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that of C18 FA.

14 15 16

KEYWORDS

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Rumen biohydrogenation, biohydrogenation intermediates, docosahexaenoic acid, in vitro.

18 19


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Introduction

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Unsaturated fatty acids (FAs) are extensively reduced to more saturated FAs in the rumen. The

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rumen biohydrogenation (RBH) process of the C18 unsaturated FAs (oleic [18:1n-9], linoleic

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[18:2n-6] and linolenic [18:3n-3] acids) and the intermediates formed have been extensively

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reviewed 1-3. The major RBH pathway of 18:2n-6 and 18:3n-3 involves a concerted enzymatic cis-

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trans isomerizations to conjugated FAs (CFA) that are subsequently reduced via proton

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abstractions, double bond migrations, and exchanges with water to form trans containing

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intermediates

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recently shown that the mixture of intermediates and metabolites formed from 18:3n-6 6 and 18:3n-

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3 7 may be even more complex than first reported.

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The long-chain polyunsaturated FAs (PUFAs) like eicosapentaenoic (EPA; 20:5n-3) and

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docoxahexaenoic (DHA; 22:6n-3) acids are also known to undergo extensive RBH as evidenced by

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the low transfer efficiency of these PUFAs into ruminant-derived products 8. However, to date only

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few studies have reported the products formed from the BH of DHA and evaluated the mechanism.

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Kairenius et al. 9 examined the products of DHA and other PUFAs in the omasal digesta from cows

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fed fish oil. They resolved many products by gas chromatography (GC) and characterized one 22:5

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product (∆5,10,13,16,19-22:5). Based on this structure and the lack of finding conjugated

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intermediates, they proposed a different pathway for the BH of DHA compared to that of C18

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PUFAs, suggesting a reduction of the cis double bond closest to the carboxyl group. Escobar et al.

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10

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intermediates of DHA. Therefore they assumed that the previously described pathway 9 was correct

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while they did not identify additional DHA metabolites. Jeyanathan et al.

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two well-known BH bacteria Butyrivibrio fibrisolvens D1 and Butyrivibrio proteoclasticus P18 for

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up to 48 h. They resolved a number of DHA products by GC and identified one major

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(∆5,10,13,16,19-22:5) and two minor 22:5 products (∆4,10,13,16,19-22:5; ∆7,10,13,16,19-22:5).

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Since they were not able to find conjugated intermediates of DHA, they supported the pathway

4,5

. With the use of isotope labeling technique and mass spectrometry (MS) it was

incubated DHA with rumen fluid from adult sheep and likewise could not find conjugated


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incubated DHA with


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reported by Kairenius et al. 9. Even though Klein and Jenkins

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DHA metabolites, they found that 13C enriched DHA was only metabolized to C22 FAs, and none

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of the C18 FAs contained the label. This put to rest the earlier speculation that DHA could be chain-

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shortened to trans-18:1 isomers that increased when DHA was included in the diet 13 or incubation

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14

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It is not the purpose of this study to investigate the effect of adding DHA on the metabolism of

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other PUFA or conditions in the rumen

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metabolism of DHA. Furthermore, it is not clear whether DHA by itself or the trans containing

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products formed during the BH of DHA, identified in this study, might affect the metabolism of

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other PUFA. For this reason it is important to actually characterize the products of DHA formed

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during the BH of DHA.

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In this manuscript we investigated the products formed during the initial stages of DHA BH using

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mixed ruminal microorganisms from sheep at conditions which were previously found to give

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appreciate levels of DHA intermediates 15. A combination of methods was used to help identify the

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products, including different highly polar GC columns, a newly developed GC- online reduction

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×GC (GC-ORxGC) method

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determine whether the RBH of DHA involved the formation of CFA as intermediates as in the case

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of the C18 PUFA, and to confirm that the metabolism of DHA does not involve chain-shortening.

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An identification of the intermediates formed during the incubation of DHA should provide a better

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understanding of how DHA is metabolized, since not all metabolites should be considered

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beneficial to health 17.

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MATERIALS AND METHODS

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Solutions

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Phosphate-bicarbonate buffer solution (PBBS) was prepared as described by Goering & Van Soest

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18

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10 µg per µL of unesterified DHA (U-84-A from Nu-Chek Prep Inc., Elysian, MN) dissolved in

did not characterize additional

.

1-3

, nor whether BH of other PUFAs will affect the

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, GC-MS and synthetic preparations. It was of particular interest to

with the exception that no trypticase was added. The concentration of the DHA solution was of


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ethanol. Methyl tricosanoate (23:0) obtained from Nu-Chek Prep Inc. was used as an internal

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standard and the concentration was 1 µg per µL in toluene.

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In vitro incubations

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Three mature sheep (3 males of Merino Branco breed) averaging 57±4.6 kg body weight were fitted

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with ruminal cannula and served as donors of rumen contents. Animals were handled and cared for

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in accordance with the EU Directive 86/609/EEC (Protocol # FMV/CEBEA 007/2016). Sheep were

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fed a maintenance diet consisting of 0.5 kg of hay and 0.5 kg of concentrate (50% barley, 20%

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sunflower meal, 17% ground corn, 10% soya meal, 1% calcium carbonate, 1% salt, 0.5% sodium

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bicarbonate, 0.5% vitatec or vitamin premix) per day for over 2 weeks in morning and afternoon

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portions. The chemical composition (g per kg of dry matter) of the concentrate and hay are 264 and

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65 for crude protein, 14 and 9.0 for total fat, 241 and 622 for neutral detergent fiber, 123 and 475

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for acid detergent fiber, and 79 and 91 for ashes, respectively.

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Between 800-1000 mL of rumen content were collected from each sheep before morning feeding,

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strained through four layers of gauze and transferred to the laboratory in three (one per animal) pre-

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warmed thermo flasks. Then, 300 mL of strained rumen fluid of each thermo were pooled together

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and kept at 39 ºC with continuous CO2 flushing. Fifty mL of the pooled and strained rumen fluid

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were mixed with 100 mL of PBBS and used in further incubations.

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Batch-cultures were performed using 15 mL Hungate tubes. The incubated substrate was composed

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by a proportional mixture of glucose, cellobiose, maltose, soluble starch, and casein, similar to other

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reports 19,20, and each tube contained 25 mg of the substrate (5 mg of each ingredient). Twenty µL

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of the DHA solution were added to each tube which gave a final DHA content of 200 µg per tube

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which was within the appropriate range to observe the formation of DHA metabolites within the

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first 6 h of incubation as previously reported 15. In order to have the same amount of ethanol in all

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tubes, 20 µL of ethanol were added to the control tubes containing no DHA. As reported by

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Morgavi et al. 21, low volumes of ethanol did not affect fermentation, and was not considered toxic

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to the rumen microorganisms. 5 ACS Paragon Plus Environment


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For incubations, volumes of 6 mL were used that were flushed with CO2, capped with a rubber

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stopper and mixed. Three tubes per time period were run under anaerobic conditions for 0, 1, 2, 3,

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and 6 h in a water bath set at 39 ºC, and tubes were individually agitated every hour. After each

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incubation time, the reaction was stopped by placing the tubes into boiling water for 5 min and

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immediately stored at -80 ºC. An exception was made for tubes at 0 h in which the appropriate

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amount of DHA was added just when the tubes were placed into the boiling water in order to avoid

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any possible reaction. Additionally, another set of tubes with no DHA were also frozen at 0 h

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(control) to determine the natural (baseline) content of DHA and other FAs in the samples. Two

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repetitions of the aforementioned design were run on separate days.

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Transesterification of rumen samples

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Samples were freeze-dried, and transesterified to FA methyl esters (FAME) in the same tube using

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a combined basic followed by acid catalysis adapted from Alves et al. 22. Briefly, 200 µL of internal

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standard solution (1 µg/µL of 23:0; methyl tricosanoate) was added to each tube containing the

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rumen fluid. Then 1.5 mL of methanol was added to each tube and sonicated for 10 min to rupture

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cells. After vortexing, tubes were placed in the sonicator for another 5 min and then 1 mL of 0.5 M

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sodium methoxide in methanol was added, vortexed and tubes were placed in a water bath at 50 ºC

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for 30 min. After cooling, 1.5 mL of 10 % HCl in methanol (v/v) was added, vortexed and heated at

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80 ºC for 15 min. After cooling, 1 mL of an aqueous solution of potassium carbonate (6%, w/w)

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was added and the methylated lipids were extracted by adding 2 mL of hexane. After mixing, the

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tubes were centrifuged at 500 g for 5 min (20 ºC). The upper hexane layer was transferred to a clean

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test tube containing approximately 1 g of anhydrous sodium sulphate. The extraction step was

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repeated with another 2 mL of hexane. The combined hexane layers were evaporated under a stream

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of N2 at 37 ºC, and the FAMEs reconstituted in about 200 µL of dichloromethane for subsequent

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purification from other constituents. Purification of the methylated lipids was performed using thin

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layer chromatography (TLC) and dichloromethane as developing solvent in order to resolve the

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FAMEs, dimethylacetals, and the FA dimethylesters and oxo-FAMEs 22. Each isolated fraction was 6 ACS Paragon Plus Environment

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then stored at -20 ºC until further analysis.

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Reduction of DHA with hydrazine

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The FAME of DHA was partially reduced with hydrazine to produce all-cis 22:5, 22:4, 22:3, 22:2

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and 22:1 FAMEs as described by Delmonte et al. 23.

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Preparation of 4,4-dimethyloxazoline (DMOX) derivatives

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Approximately 1-5 mg of the BH products as FAME were hydrolyzed to free FAs (FFA) by

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reaction with 1 mL of 0.1 M KOH in 90% ethanol, at 50°C for 2 hours. The solution was acidified

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with 0.5 mL of 1 M HCl. After addition of 5 mL of water and 3 mL of hexane:diethylether (1:1),

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the FFA were recovered. The FFAs were reduced to dryness at the bottom of a 1 mL conical screw

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cap reaction vial, and then 0.5 mL of 2-amino-2-methyl-1-propanol was added. The container was

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purged with argon and heated for 2 h at 190 ºC 24. The reaction products were transferred into a 20

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mL test tube containing 5 mL of hexane:diethylether (1:1) and 5 mL of water. The test tube was

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gently mixed and let rest until the layers separated. The organic layer was washed with another 5

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mL of water, passed through 500 mg of sodium sulfate, and concentrated to 50 µL with a stream of

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nitrogen.

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GC-FID analysis

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The FAMEs were analyzed using a gas chromatograph (HP 6890A; Hewlett-Packard, Avondale,

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PA) equipped with a fame-ionization detector (GC-FID) and a Supelco SP-2560 capillary column

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(100 m x 0.25 mm i.d. x 0.20 µm film thickness; Bellefonte, PA). The injector and detector

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temperatures were set at 250 ºC and 280 ºC, respectively. The FAME samples were analyzed by

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applying a temperature program with a plateau at 175 ºC and at 150 ºC to enhance the resolution of

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the trans-18:1 isomers as previously described 25. The equivalent chain lengths (ECL) values of

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selected FAMEs from the incubation mixtures and bovine milk were calculated 26 from separations

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acquired using an isothermal temperature at 200 ºC. Hydrogen was used as carrier gas at a flow rate

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of 1 mL / min, and 1 µL of sample was injected with a split ratio of 50:1. For FAME identification,

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reference standards #463 and #603, plus individual FAMEs of 21:0, 23:0, 26:0, 28:0 and CLA 7 ACS Paragon Plus Environment


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mixture #UC-59M were used (Nu-Chek Prep Inc., Elysian, MN). Branched-chain FAs were

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identified using a bacterial FAME mixture purchased from Matreya (Pleasant Gap, PA).

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Selected samples were also analyzed using the Supelco SLB-IL111 ionic capillary column (200 m x

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0.25 mm i.d. x 0.20 µm film thickness; Bellefonte, PA) 27,28. In this case, the injector and detector

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temperatures were set at 300 ºC and 250 ºC, respectively. Initial oven temperature of 170 ºC was

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held for 50 min, increased at 6 ºC / min to 185 ºC and held for 35 min. Hydrogen was used as

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carrier gas at a flow rate of 1.6 mL / min for 35 min, then increased 0.3 mL / min / min to a final

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flow rate of 3 mL / min. One µL of sample was injected and the split ratio was 100:1.

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GC-online reduction × GC (GC-OR×GC) analysis

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The GC-OR×GC separations were acquired by using the instrumental configuration and conditions

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previously described with minor modifications 16. The apparatus consisted of an Agilent 7890A GC-

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FID (Wilmington, DE) equipped with a dual stage thermal modulator Zoex ZX2 (Houston, TX),

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and a split/splitless injection port. The column set was sequentially composed of a SLB-IL111

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capillary column (100 m x 0.25 mm i.d. x 0.25 µm film thickness; Supelco, Inc., Bellefonte, PA), a

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0.20 m x 0.18 mm i.d. capillary tube coated with palladium, a 2 m x 0.10 mm deactivated uncoated

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capillary tube (modulator loop), and a SLB-IL111 capillary column (1 m x 0.10 mm i.d., 0.08 mm

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film thickness; Supelco). The modulation spots were set at 0.05 m after the beginning of the

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modulation loop and 0.05 m from the beginning of the second separation column. The modulation

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time was set to 8 sec. The oven was maintained at the constant temperature of 170 ºC, and the

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injector port at 300 ºC. The FID was maintained at 250 ºC and fed with 30 mL / min of N2 make up

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gas, 400 mL / min air, and 30 mL / min of H2. Hydrogen was used as carrier gas at a constant

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pressure of 60 psi. The split flow was set to 26.88 mL / min, and the injection volume to 1 µL. The

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acquisition rate was set to 200 hz, and data were processed with the GC Image GC×GC software

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(Version 2.1, GC Image, LLC, Lincoln, Nebraska). All capillary connections were made with Micro

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Unions (SGE Analytical Science, Australia).

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GC-MS separation of FAMEs in the CI+ mode 8 ACS Paragon Plus Environment

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GC-MS separations in the chemical ionisation (CI+) mode were performed with an Agilent 7200A

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gas chromatography quadrupole time-of-flight mass spectrometry (GC-QTOF-MS), equipped with

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a SP-2560 capillary column (100 m x 0.25 mm, 0.20 µm film thickness) followed by a purged union

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and a 2 m x 0.10 mm deactivated retention gap connecting to the MS. The carrier gas was He at the

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constant flow of 1.1 mL / min increased to 1.2 mL / min after the purged union. The oven

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temperature was maintained at 45 ºC for 4 min, ramped to 175 ºC at 13 ºC / min, maintained for 27

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min, then ramped at 4 ºC / min to 225 ºC and maintained for 100 min. The injection volume was 1

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µL in splitless mode, and the injection port was purged after 2 min with 70 mL / min of carrier gas.

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The injection port and transfer line temperature was set to 250 ºC, and the ion source to 300 ºC. The

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MS was operated in CI+ mode with NH3 as chemical ionization reagent. The filament current was

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set to 100 µA.

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GC-MS separation of DMOX derivative in the EI+ mode

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Experimental conditions were the same as for the CI+ analyses of FAMEs with the following

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exceptions. The oven temperature was maintained at 190 ºC for the entire separation (200 min), 1

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µL was injected in split mode with a split ratio of 1:15. The MS was operated in EI+ mode with the

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electron energy of 50 eV and the filament current of 100 µA.

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Statistical analysis

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Data were analyzed using the PROC MIXED from SAS 9.4 29 considering the incubation run as

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random block and the incubation time as a fixed categorical effect. When significant effects

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(P 0.05).

759 760 761

Figure 10. Proposed pathways of DHA metabolism using sheep rumen fluid. One of the five

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possible MTMI-DHA and two of the MC-DHA metabolites are shown as representative products,

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as well as the five 22:5 products identified, all of which contain an isolated trans double bond. (*)

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Members of the pathway of DHA metabolism that resulted in the formation of the major 22:5

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product in the incubation mixture. (**) Product reported by Jeyanathan et al.(11) that could not be

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confirmed.

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MTMI-DHA, mono trans methylene interrupted-DHA; MC-DHA, mono-conjugated DHA.

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Table 1. Products of DHA incubation with sheep mixed rumen fluid for 0, 1, 2, 3, and 6 hours.

µg / tube 22:6 n-3 (DHA) MC-DHA MTMI-DHA 22:x unidentified sum of DHA products DHA + DHA products 22:5 22:4 22:3 22:2 22:1 cis-11 and cis-13 22:0

0 190a 0.00c 0.91c 9.20d 10.1d 200a 0.88c 0.01d 0.89b 1.01 1.29 12.7bc

Incubation time (h) 1 2 b 146 102c 5.96b 9.70a b 8.53 11.46a c 31.9 54.9b c 46.4 76.1b 192ab 178b bc 1.27 1.93ab c 0.52 0.76c a 1.19 1.37a 1.10 0.92 1.11 1.34 12.5c 13.0ab

3 83.3d 9.91a 11.6a 69.6b 91.1b 174bc 2.37a 1.10b 1.23a 1.16 1.36 12.9abc

6 36.2e 7.61ab 9.49b 102a 119a 156c 2.22a 1.74a 1.40a 1.44 1.20 13.1a

SEM 3.88 1.07 2.16 6.75 7.07 6.93 0.36 0.13 0.30 0.18 0.07 0.62

P-value

Evidence for the Initial Steps of DHA Biohydrogenation by Mixed

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