Novel characterisation of minor α-linolenic acid isomers in linseed oil by gas chromatography and covalent adduct chemical ionisation tandem mass spectrometry.

Discrimination between polyunsaturated fatty acid isomers with three double bonds is a great challenge, due to structural similarities and similar polarities. In this study, we report the identification of four minor geometrical isomers of α-linolenic acid (ALA) present in linseed oil samples: (9E,12Z,15E)-, (9Z,12Z,15E)-, (9Z,12E,15Z)- and (9E,12Z,15Z)-octadeca-9,12,15-trienoic acids, chromatographically resolved by gas chromatography (GC) using a new and highly polar ionic phase column (SLB-IL111). Gas chromatography-electron ionisation mass spectrometry (GC-EIMS) determined that the four unknown compounds were C18:3 n-3 isomers. The positional 9-12-15 C18:3 configuration was achieved by covalent adduct chemical ionisation tandem mass spectrometry (CACI-MS/MS) while geometrical configuration was established with analytical standards based on relative retention. We hypothesised that these isomers are formed during linseed oil deodorisation and postulate preferred and unfavoured isomerisation pathways of ALA.

Imbalance of folic acid and vitamin B12 is associated with birth outcome: an Indian pregnant women study.

BACKGROUND/OBJECTIVES: Maternal nutrient supplementation in developing countries is generally restricted to provision of iron and folic acid. Along with folic acid, vitamin B12 is also an important determinant of fetal growth and development. During pregnancy, the increased requirement of folic acid is met with supplementation, while vitamin B12 remains untreated and possibly deficient. The objective of our study was to study the combined effect of maternal plasma folate and vitamin B12, and their ratio on birth anthropometrics. SUBJECTS/METHODS: We carried out an observational study on 49 full-term pregnant women at KEM Hospital, Pune, India, during 2006-2008, and measured plasma folate, vitamin B12 and homocysteine in venous blood at 36 weeks of gestation. Neonatal anthropometrics (birth weight, length, head circumference, abdominal circumference, mid arm circumference, chest circumference, triceps skinfold and subscapular skinfold thickness) were measured within 24 h of birth. RESULTS: Maternal plasma folate and vitamin B12 were not correlated to neonatal anthropometrics. The combined association of folate and vitamin B12 expressed as folate to vitamin B12 ratio was correlated to the neonatal anthropometrics. Imbalance in the maternal micronutrients with increasing ratio of folate to vitamin B12 was associated with an increase in plasma homocysteine (P=0.014), lowering of neonatal birth weight (P=0.009), birth length (P=0.034), head circumference (P=0.018) and chest circumference (P=0.009), while no significant association to other anthropometrics was observed. CONCLUSIONS: Supplementation of vitamin B12 in addition to supplementation of folic acid in pregnancy may be important for improving birth weight, birth length, head circumference and chest circumference.

The effects of EPA, DHA, and aspirin ingestion on plasma lysophospholipids and autotaxin.

Lysophophatidylcholine (LPC) and lysophosphatidic acid (LPA) are potent lysolipid mediators increasingly linked with atherosclerosis and inflammation. A current model proposing that plasma LPA is produced when LPC is hydrolyzed by the enzyme autotaxin has not been rigorously investigated in human subjects. We conducted a clinical trial of eicosapentaenoic acid/docosahexaenoic acid (EPA/DHA) and aspirin ingestion in normal volunteers. Fasting blood samples were drawn at baseline and after 4-week supplementation with EPA/DHA (3.4 g/d) with and without aspirin (650 mg). Plasma LPC and LPA species and autotaxin activity were measured. EPA-LPC and DHA-LPC concentrations increased significantly with EPA/DHA supplementation whereas EPA- and DHA-LPA did not. Autotaxin activity was unaffected by any treatment, and aspirin had no effect on any endpoint. Taken together, our data demonstrate that plasma LPC, but not LPA, species can be dynamically regulated by dietary supplementation, and argue against a simple model of LPA generation via LPC hydrolysis.

Milk fat synthesis is unaffected by abomasal infusion of the conjugated diene 18:3 isomers cis-6,trans-10, cis-12 and cis-6,trans-8,cis-12.

It has been previously established that trans-10,cis-12 CLA is a potent inhibitor of milk fat synthesis. Although the mechanism of this action is not completely understood, it has been speculated that eicosanoid-like metabolites of this isomer formed by the activity of tissue desaturases may be responsible for its activity. The objective of this study was to investigate the effects of an enrichment containing an 18:3 conjugated diene, produced in the metabolism of trans-10,cis-12 CLA, on milk fat synthesis. Three rumen-fistulated Holstein cows (210+/-8 d in milk) were randomly assigned in a 3 x 3 Latin square experiment. Treatments were (i) control, (ii) trans-10,cis-12 CLA supplement (2.1 g/d; positive control), (iii) enrichment providing two conjugated diene 18:3 isomers (2.6 g/d of cis-6,trans-10,cis-12 and 4.0 g/d of cis-6,trans-8,cis-12) and trans-10,cis-12 CLA (2.1 g/d). Treatments were abomasally infused for 5 d at 4-h intervals, and there was a 7-d interval between periods. Milk yield, dry matter intake, and milk protein yield were unaffected by treatments. In contrast, the trans-10,cis-12 CLA supplement reduced milk fat yield by 27%, whereas the supplement enriched with conjugated diene 18:3 isomers (treatment iii) had no effect on milk fat yield beyond that attributable to its trans-10,cis-12 CLA content. The transfer efficiency of trans-10,cis-12 CLA into milk fat was 25 and 24% for treatments ii and iii, respectively. At the same time, the abomasally infused conjugated diene 18:3 isomers were transferred to milk fat with an efficiency of 33 and 41% for cis-6,trans-10,cis-12 and cis-6,trans-8,cis-12 18:3, respectively. Overall, short-term abomasal infusion of the conjugated diene 18:3 isomers had no effect on milk fat synthesis, thereby offering no support for an involvement of metabolites of trans-10,cis-12 CLA in the regulation of milk fat synthesis.

Significant utilization of dietary arachidonic acid is for brain adrenic acid in baboon neonates.

Dietary arachidonic acid (20:4n-6) utilization in-vivo for carbon recycling into de-novo lipogenesis and conversion to n-6 long chain polyunsaturates was investigated in baboon neonates using [U-(13)C]20:4n-6. Neonates consuming a formula typical of human milk received a single oral dose of [(13)C]arachidonic acid in sn-2 position of either triglyceride or phosphatidylcholine at 18-19 days of postnatal life. Neonate brain, retina, liver, and plasma were obtained 10 days later (28-29 days of life). Low isotopic enrichment (0.27-1.0%Total label) was detected in dihomo-gamma-linolenic acid (20:3n-6) in all tissues, but label incorporation into saturates or monounsaturates was not detected. In neonate brain and retina, 16% and 11% of total label was recovered in 22:4n-6, respectively. The relative contribution of dietary fatty acids to postnatal brain 22:4n-6 accretion can be estimated for dietary 20:4n-6 and preformed 22:4n-6 as 17% and 8%, respectively, corresponding to efficiencies of 0.48% and 0.54% of dietary levels, respectively. These results demonstrate in term baboon neonates that in vivo 1) 20:4n-6 was retroconverted to 20:3n-6, 2) 20:4n-6 did not contribute significantly to de novo lipogenesis of saturates and monounsaturates, and 3) the preformed 20:4n-6 contribution to brain 22:4n-6 accumulation was quantitatively a significant metabolic fate for dietary 20:4n-6.

High-precision isotope ratio mass spectrometry and stable isotope precursors for tracer studies in cell culture.

The use of stable isotope-labeled tracers is demonstrated in an in vitro system with analysis by high-precision isotope ratio mass spectrometry (IRMS), using n-3 long-chain polyunsaturated fatty acid (LCP) biosynthesis from [U-(13)C]18:3n-3 (18:3n-3*) in Y79 human retinoblastoma cells as a model system. The cells were cultured as a suspension in RPMI 1640 medium supplemented with 15% fetal calf serum at 37 degrees C with 5% CO(2) in air. They were harvested by sedimentation and cell lipids were extracted to determine the presence of 18:3n-3* metabolites using gas chromatography-combustion (GCC)-IRMS. As the dose of 18:3n-3* was systematically increased from treatment to treatment, the atom percent excess and the amounts of biosynthesized LCP* increased, while the percentage dose in each n-3 LCP* remained constant. Cultures incubated with 0.5 micromol (10 microM) of albumin-bound 18:3n-3, composed of 18:3n-3* diluted 1/60 or 1/100 with natural abundance 18:3n-3, yielded products with enrichments about 1.5 at.% excess (delta(13)C(PDB) < 1500 per thousand), which is optimal for high-precision measurements. Kinetics in Y79 cells incubated with 18:3n-3* showed that n-3 LCP* incorporation increased over time; 18:3n-3*, 20:5n-3*, 22:5n-3*, and 22:6n-3* were detected at all time points with the 1/60 dilution. These data document experimental parameters for optimal stable isotope use and IRMS detection for in vitro tracer methodology.

Dietary 18:3n-3 and 22:6n-3 as sources of 22:6n-3 accretion in neonatal baboon brain and associated organs.

The bioequivalence of dietary linolenic acid (LNA) and docosahexaenoic acid (DHA) for brain DHA accretion was measured in neonatal baboons at 4-6 wk of age using stable isotope tracers. Neonates consumed a conventional U.S. term-infant formula devoid of long chain polyunsaturates and with an n-6/n-3 ratio of about 10:1. At 4 wk of age, neonates were dosed with either 13C LNA or 13C DHA. At 6 wk of age, neonate brain, retina, and other organs were harvested for fatty acid and isotopic analyses. The relative accretion of labeled DHA was 7-fold greater as a percentage of dose for the DHA-dosed animals compared to the LNA-dosed animals. The baboon is an omnivore that regularly consumes meat and insects; its plasma lipid profile responds similarly to humans in response to changes in feeding and living habits. These observations suggest that the baboon is a suitable model for human unsaturated fatty acid studies.

Delay of preterm delivery in sheep by omega-3 long-chain polyunsaturates.

A positive correlation has been shown between dietary intake of long-chain omega-3 fatty acids in late pregnancy and gestation length in pregnant women and experimental animals. To determine whether omega-3 fatty acids have an effect on preterm labor in sheep, a fish oil concentrate emulsion was continuously infused to six pregnant ewes from 124 days gestational age. At 125 days, betamethasone was administered to the fetus to produce preterm labor. Both the onset of labor and the time of delivery were delayed by the fish oil emulsion. Two of the omega-3-infused ewes reverted from contractions to nonlabor, an effect never previously observed for experimental glucocorticoid-induced preterm labor in sheep. Maternal plasma estradiol and maternal and fetal prostaglandin E2 rose in control ewes but not in those infused with omega-3 fatty acid. The ability of omega-3 fatty acids to delay premature delivery in sheep indicates their possible use as tocolytics in humans. Premature labor is the major cause of neonatal death and long-term disability, and these studies present information that may lead to a novel therapeutic regimen for the prevention of preterm delivery in human pregnancy.

Increasing dietary linoleic acid in young rats increases and then decreases docosahexaenoic acid in retina but not in brain.

The accumulation of fatty acids in retina, brain, liver, and plasma of 30-day-old rat pups consuming various levels of linoleic acid (LA, 18:2n-6) and constant alpha-linolenic acid (ALA, 18:3n-3) is reported. Dams were fed graded levels of LA during gestation and lactation, and the pups were maintained on the diet of their dams until the end of the brain growth spurt at 30 d of life. Milk, and pup brain, retina, liver, and plasma were analyzed quantitatively for fatty acid profile. The percentage of docosahexaenoic acid (DHA, 22:6n-3) in retina increased from an LA-deficient dietary level, peaked at the 9:1 (LA/ALA) level, then fell for the 41:1 and 69:1 levels. In contrast, the brain DHA percentage was unaffected by dietary LA levels. Retinal unsaturated fatty acid levels paralleled liver and plasma levels. The milk fatty acid composition mirrored the diets. These data show that the retinal fatty acid composition responds sensitively to dietary fatty acid composition, similar to liver and plasma, while the brain unsaturate composition is nearly independent of dietary composition.