Cynthia Tyburczy

An alternate pathway to long-chain polyunsaturates: the FADS2 gene product Δ8-desaturates 20:2n-6 and 20:3n-3

The mammalian Delta6-desaturase coded by fatty acid desaturase 2 (FADS2; HSA11q12-q13.1) catalyzes the first and rate-limiting step for the biosynthesis of long-chain polyunsaturated fatty acids. FADS2 is known to act on at least five substrates, and we hypothesized that the FADS2 gene product would have Delta8-desaturase activity. Saccharomyces cerevisiae transformed with a FADS2 construct from baboon neonate liver cDNA gained the function to desaturate 11,14-eicosadienoic acid (20:2n-6) and 11,14,17-eicosatrienoic acid (20:3n-3) to yield 20:3n-6 and 20:4n-3, respectively. Competition experiments indicate that Delta8-desaturation favors activity toward 20:3n-3 over 20:2n-6 by 3-fold. Similar experiments show that Delta6-desaturase activity is favored over Delta8-desaturase activity by 7-fold and 23-fold for n-6 (18:2n-6 vs 20:2n-6) and n-3 (18:3n-3 vs 20:3n-3), respectively. In mammals, 20:3n-6 is the immediate precursor of prostaglandin E1 and thromboxane B1. 20:3n-6 and 20:4n-3 are also immediate precursors of long-chain polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid, respectively. These findings provide unequivocal molecular evidence for a novel alternative biosynthetic route to long-chain polyunsaturated fatty acids in mammals from substrates previously considered to be dead-end products.

Characterization of cis-9 trans-11 trans-15 C18:3 in milk fat by GC and covalent adduct chemical ionization tandem MS

Rumen biohydrogenation of dietary α-linolenic acid gives rise in ruminants to accumulation of fatty acid intermediates, some of which may be transferred into milk. Rumelenic acid [cis-9 trans-11 cis-15 C18:3 (RLnA)] has recently been characterized, but other C18:3 minor isomers are still unknown. The objective of this work was to identify a new isomer of octatridecenoic acid present in milk fat from ewes fed different sources of α-linolenic acid. Structural characterization of this fatty acid was achieved by GC-MS. Analysis of dimethyloxazoline and picolinyl ester derivatives allowed for location of the double bond positions. Covalent adduct chemical ionization tandem mass spectrometry confirmed the positional structure 9-11-15, identical to RLnA, and helped to establish double bond geometry (cis-trans-trans). This new C18:3 isomer could be formed by isomerization of cis-15 bond of RLnA and subsequently converted by hydrogenation to trans-11 trans-15 C18:2, an octadecadienoic acid also detected in this study.

Uptake and Utilization of Trans Octadecenoic Acids in Lactating Dairy Cows

Trans fatty acids (FA) arise in ruminant-derived foods as a consequence of rumen biohydrogenation and are of interest because of their biological effects and potential role in chronic human diseases. Our objective was to compare 2 trans FA, elaidic acid (EA; trans-9 18:1) and vaccenic acid (VA; trans-11 18:1), with oleic acid (OA; cis-9 18:1) relative to plasma lipid transport and mammary utilization for milk fat synthesis. Three ruminally cannulated, Holstein dairy cows, 259 +/- 6 DIM (mean +/- SEM), were randomly assigned in a 3 x 3 Latin square design. Treatments were a 4-d abomasal infusion of 1) OA (45.5 g/d), 2) EA (41.7 g/d), and 3) VA (41.4 g/d). Milk samples were collected at each milking and blood samples were collected at the start and end of each treatment period. The proportions of total plasma FA associated with each plasma lipid fraction at baseline (pretreatment) were 62.6 +/- 0.6% phospholipids, 26.1 +/- 0.6% cholesterol esters, 9.8 +/- 0.4% triglycerides, and 1.5 +/- 0.1% nonesterified fatty acids; these values were unaffected by treatment. There were striking differences in the FA composition of the individual plasma lipid fractions and in the distribution of specific 18-carbon FA among the lipid fractions. Infusion of treatment isomers caused their specific increase in the various plasma lipid fractions but had no effect on milk production variables, including milk fat yield and content. Transfer efficiency of infused OA, EA, and VA to milk fat averaged 65.5 +/- 3.0%, 59.7 +/- 1.5%, and 54.3 +/- 0.6%, respectively. For the VA infusion, 24.6 +/- 1.1% of the transfer was accounted for by the increased yield of cis-9, trans-11 conjugated linoleic acid in milk fat, consistent with its endogenous synthesis from VA via the mammary enzyme Delta(9)-desaturase. Notably, linoleic acid (18:2n-6) and linolenic acid (18:3n-3) accounted for 47.7% of total plasma FA, but only 2.6% of FA in milk. Overall, results demonstrate clear differences in plasma transport and mammary uptake and utilization of 18-carbon FA, and these relate to the location, orientation, and number of double bonds.

Plasma oxylipin profiling identifies polyunsaturated vicinal diols as responsive to arachidonic acid and docosahexaenoic acid intake in growing piglets

The dose-responsiveness of plasma oxylipins to incremental dietary intake of arachidonic acid (20:4n-6; ARA) and docosahexaenoic acid (22:6n-3; DHA) was determined in piglets. Piglets randomly received one of six formulas (n = 8 per group) from days 3 to 27 postnatally. Diets contained incremental ARA or incremental DHA levels as follows (% fatty acid, ARA/DHA): (A1) 0.1/1.0; (A2) 0.53/1.0; (A3–D3) 0.69/1.0; (A4) 1.1/1.0; (D1) 0.66/0.33; and (D2) 0.67/0.62, resulting in incremental intake (g/kg BW/day) of ARA: 0.07 ± 0.01, 0.43 ± 0.03, 0.55 ± 0.03, and 0.82 ± 0.05 at constant DHA intake (0.82 ± 0.05), or incremental intake of DHA: 0.27 ± 0.02, 0.49 ± 0.03, and 0.81 ± 0.05 at constant ARA intake (0.54 ± 0.04). Plasma oxylipin concentrations and free plasma PUFA levels were determined at day 28 using LC-MS/MS. Incremental dietary ARA intake dose-dependently increased plasma ARA levels. In parallel, ARA intake dose-dependently increased ARA-derived diols 5,6- and 14,15-dihydroxyeicosatrienoic acid (DiHETrE) and linoleic acid-derived 12,13-dihydroxyoctadecenoic acid (DiHOME), downstream metabolites of cytochrome P450 expoxygenase (CYP). The ARA epoxide products from CYP are important in vascular homeostatic maintenance. Incremental DHA intake increased plasma DHA and most markedly raised the eicosapentaenoic acid (EPA) metabolite 17,18-dihydroxyeicosatetraenoic acid (DiHETE) and the DHA metabolite 19,20-dihydroxydocosapentaenoic acid (DiHDPE). In conclusion, increasing ARA and DHA intake dose-dependently influenced endogenous n-6 and n-3 oxylipin plasma concentrations in growing piglets, although the biological relevance of these findings remains to be determined.

Heart arachidonic acid is uniquely sensitive to dietary arachidonic acid and docosahexaenoic acid content in domestic piglets.

This study determined the sensitivity of heart and brain arachidonic acid (ARA) and docosahexaenoic acid (DHA) to the dietary ARA level in a dose-response design with constant, high DHA in neonatal piglets. On day 3 of age, pigs were assigned to 1 of 6 dietary formulas varying in ARA/DHA as follows (% fatty acid, FA/FA): (A1) 0.1/1.0; (A2) 0.53/1.0; (A3-D3) 0.69/1.0; (A4) 1.1/1.0; (D2) 0.67/0.62; and (D1) 0.66/0.33. At necropsy (day 28) higher levels of dietary ARA were associated with increased heart and liver ARA, while brain ARA remained unaffected. Dietary ARA had no effect on tissue DHA accretion. Heart was particularly sensitive, with pigs in the intermediate groups having different ARA (A2, 18.6±0.7%; A3, 19.4±1.0%) and a 0.17% increase in dietary ARA resulted in a 0.84% increase in heart ARA. Further investigations are warranted to determine the clinical significance of heart ARA status in developing neonates.

Evaluation of bioequivalency and toxicological effects of three sources of arachidonic acid (ARA) in domestic piglets.

Arachidonic acid (ARA) and docosahexaenoic acid (DHA) are routinely added to infant formula to support growth and development. We evaluated the bioequivalence and safety of three ARA-rich oils for potential use in infant formula using the neonatal pig model. The primary outcome for bioequivalence was brain accretion of ARA and DHA. Days 3-22 of age, domestic pigs were fed one of three formulas, each containing ARA at ∼0.64% and DHA at ∼0.34% total fatty acids (FA). Control diet ARA was provided by ARASCO and all diets had DHA from DHASCO (Martek Biosciences Corp., Columbia, MD). The experimental diets a1 and a2 provided ARA from Refined Arachidonic acid-rich Oil (RAO; Cargill, Inc., Wuhan, China) and SUNTGA40S (Nissui, Nippon Suisan Kaisha, Ltd., Tokyo, Japan), respectively. Formula intake and growth were similar across all diets, and ARA was bioequivalent across treatments in the brain, retina, heart, liver and day 21 RBC. DHA levels in the brain, retina and heart were unaffected by diet. Liver sections, clinical chemistry, and hematological parameters were normal. We conclude that RAO and SUNTGA40S, when added to formula to supply ∼0.64% ARA are safe and nutritionally bioequivalent to ARASCO in domestic piglets.

Growth, clinical chemistry and immune function in domestic piglets fed varying ratios of arachidonic acid and DHA

In the USA, infant formulas contain long-chain PUFA arachidonic acid (ARA) and DHA in a ratio of 2:1 and comprise roughly 0·66 g/100 g and 0·33 g/100 g total fatty acids (FA). Higher levels of dietary DHA appear to provide some advantages in visual or cognitive performance. The present study evaluated the effect of physiologically high dietary ARA on growth, clinical chemistry, haematology and immune function when DHA is 1·0 g/100 g total FA. On day 3 of age, formula-reared (FR) piglets were matched for weight and assigned to one of six milk replacer formulas. Diets varied in the ratio of ARA:DHA as follows (g/100 g FA/FA): A1, 0·1/1·0; A2, 0·53/1·0; A3-D3, 0·69/1·0; A4, 1·1/1·0; D2, 0·67/0·62; D1, 0·66/0·33. A seventh group was maternal-reared (MR) and remained with the dam during the study. Blood collection and body weight measurements were performed weekly, and piglets were killed on day 28 of age. No significant differences were found among any of the FR groups for formula intake, growth, clinical chemistry, haematology or immune status measurements. A few differences in clinical chemistry, haematology and immune function parameters between the MR pigs and the FR groups probably reflected a difference in growth rate. We conclude that the dietary ARA level up to 1·0 g/100 g total FA is safe and has no adverse effect on any of the safety outcomes measured, and confirm that DHA has no adverse effect when ARA is at 0·66 g/100 g FA.

Acetonitrile covalent adduct chemical ionization tandem mass spectrometry of non‐methylene‐interrupted pentaene fatty acid methyl esters

Acetonitrile covalent adduct chemical ionization tandem mass spectrometry (CACIMS/MS) has shown to be an efficient method for the identification of double-bond position in homoallylic, conjugated and several polyene non-methylene-interrupted (NMI) fatty acid methyl esters. However, it has not been thoroughly evaluated for NMI highly unsaturated fatty acids (HUFA) with more than four double bonds. Docosahexaenoic acid (DHA)-rich single cell oil (DHASCO(®); Martek Biosciences, Corp.) was partially hydrogenated (partially hydrogenated DHASCO; PHDO) producing ten novel 22:5 and 22:6 HUFA isomers. In single-stage MS, the ratio of [M+54](+)/[M+54-32](+) for the 22:5 and 22:6 isomers indicated the presence of homoallylic or partially conjugated double-bond systems. The CACIMS/MS spectra revealed six 22:5 isomers with diagnostic ions corresponding to the homoallylic 22:5n-6 and 22:5n-3 isomers, and four distinct NMI 22:5 isomers. Diagnostic ions for four 22:6 isomers were identical to the native DHA illustrating that CACIMS/MS is sensitive to double-bond position but not geometry. Three gas chromatography (GC) peaks for partially conjugated 22:6 isomers were also detected and clearly distinguishable from homoallylic 22:6 isomers, but their CACIMS/MS spectra did not yield prominent ions indicative of double-bond position, possibly due to co-elution. Overall, CACIMS/MS was effective for determining double-bond position in NMI 22:5 isomers. Further investigations are warranted to evaluate and determine fragmentation patterns for partially conjugated and NMI 22:6 HUFA.