A rare eicosanoid precursor analogue, sciadonic acid (5Z,11Z,14Z-20:3), detected in vivo in hormone positive breast cancer tissue.

Numerous genetic alterations of HSA 11q13 are found frequently in several cancer types, including breast cancer (BC). The 11q13 locus harbors FADS2 encoding Δ6 desaturation which is not functional in several cancer cell lines, including hormone positive MCF7 BC cells. In vitro, the non-functional FADS2 activity unmasks 18:2n-6 elongation to 20:2n-6 and Δ5 desaturation by FADS1 to yield 5Z,11Z,14Z-20:3 (sciadonic acid) rather than 5Z,8Z,11Z,14Z-20:4 (arachidonic acid). In this pilot study we aimed to determine whether 5,11,14-20:3 appears in vivo in hormone positive human BC tissue. Fatty acids were profiled in surgically removed human breast tumor and adjacent normal tissue (n = 9). Sciadonic acid was detected in three of nine breast tumor samples and was below detect limits in normal breast tissue. The internal Δ8 double bond of arachidonic acid is required for normal eicosanoid synthesis but is missing in sciadonic acid. This pilot study demonstrates for the first time in vivo sciadonic acid in hormone positive BC tissue, warranting a larger survey study to further evaluate its appearance and the functional implications.

BCFA suppresses LPS induced IL-8 mRNA expression in human intestinal epithelial cells.

Branched chain fatty acids (BCFA) are components of common food fats and are major constituents of the normal term human newborn GI tract. Polyunsaturated fatty acids (PUFA) have been suggested to reduce the risk and development of inflammatory bowel diseases (IBD); however, little is known about the influence of BCFA on inflammation. We investigated the effect of BCFA on interleukin (IL)-8 and NF-κB production in a human intestinal epithelial cell line (Caco-2). Cells were pre-treated with specific BCFA, or DHA, or EPA, and then activated with lipopolysaccharide (LPS). Both anteiso- and iso- BCFA reduce IL-8. Anteiso-BCFA more effectively suppressed IL-8 than iso-BCFA in LPS stimulated Caco-2 cells. However BCFA in general were less effective than DHA or EPA. Activated BCFA-treated cells expressed less of the cell surface Toll-like receptor 4 (TLR-4) compared to controls. These are the first data to show the reduction of pro-inflammatory markers in human cells mediated by BCFA.

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.

Long-chain polyunsaturated fatty acids attenuate the IL-1ß-induced proinflammatory response in human fetal intestinal epithelial cells.

BACKGROUND: Evidence suggests that excessive inflammation of the immature intestine may predispose premature infants to necrotizing enterocolitis (NEC). We investigated the anti-inflammatory effects of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (ARA) in human fetal and adult intestinal epithelial cells (IEC) in primary culture. METHODS: Human fetal IEC in culture were derived from a healthy fetal small intestine (H4) or resected small intestine of a neonate with NEC (NEC-IEC). Intestinal cell lines Caco2 and NCM460 in culture were used as models for mature IEC. IEC in culture were pretreated with 100 µmol/l palmitic acid (PAL), DHA, EPA, ARA, or ARA+DHA for 48 h and then stimulated with proinflammatory IL-1ß. RESULTS: DHA significantly attenuated IL-1ß induced proinflammatory IL-8 and IL-6 protein and mRNA in fetal H4, NEC-IEC, and mature Caco2, NCM460 IEC, compared to control and PAL treatment. DHA downregulated IL-1R1 (IL-1ß receptor) and NFk ß1 mRNA expression in fetal and adult IEC. ARA had potent anti-inflammatory effects with lower IL-8 and IL-6 (protein and mRNA) in fetal H4 but not in NEC-IEC or adult IEC. CONCLUSION: The present study provides evidence that DHA and ARA may have important anti-inflammatory functions for prevention of NEC in premature infants.

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.

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.

Maternal intravenous administration of long chain n-3 polyunsaturates to the pregnant ewe in late gestation results in specific inhibition of prostaglandin h synthase (PGHS) 2, but not PGHS1 and oxytocin receptor mRNA in myometrium during betamethasone-induced labor.

OBJECTIVES: Both the onset of labor and time to delivery during betamethasone-induced delivery are delayed by omega-3 polyunsaturated fatty acid (PUFA) administration to pregnant sheep. That fatty acid also inhibits the labor-related increase in maternal plasma estradiol and maternal and fetal prostaglandin E(2). To evaluate the mechanism of inhibition of prostaglandin production and delay of onset of labor and time of delivery in PUFA-treated sheep, we determined the effect of PUFA on myometrial prostaglandin H synthase (PGHS) 1 and 2 and oxytocin receptor mRNA levels in betamethasone-induced labor. METHODS: At 124 days' gestation, a 20% emulsion of either intralipid (IL, n = 6) or PUFA (n = 6) was infused continuously (3 mL/kg per day) intravenously (IV) to the ewe. At 125 days' gestation, betamethasone was administered IV (10 microg/h over 48 hours) to fetuses of both intralipid- and PUFA-treated ewes. Myometrium was collected at necropsy either during betamethasone-induced labor as evaluated by myometrial electromyography or within 5 days of the termination of betamethasone infusion, if delivery did not occur after fetal betamethasone infusion. Total myometrial RNA was analyzed by Northern blot for oxytocin receptor and PGHS1 and 2 mRNA normalized for 18s. RESULTS: Treatment with PUFA decreased myometrial PGHS2 mRNA but did not alter myometrial PGHS1 and oxytocin receptor mRNA after betamethasone administration. CONCLUSIONS: This finding provides a mechanism whereby PUFA delays betamethasone-induced delivery in sheep and suggests a potential role of PUFA as an effective tocolytic agent in human pregnancy.

An octaene fatty acid, 4,7,10,13,16,19,22,25-octacosaoctaenoic acid (28:8n-3), found in marine oils.

We report structure determination of an octaene fatty acid, 4,7,10, 13,16,19,22,25-octacosaoctaenoic acid (28:8n-3). The molecular weight and double bond locations were determined using acetonitrile chemical ionization mass spectrometry (MS) and MS/MS and were confirmed by MS of hydrogenated and deuterogenated 28:8 and by argentation thin-layer chromatography. 28:8n-3 was 1.2 +/- 0.1%, in oil derived from the heterotrophic dinoflagellate Crypthecodinium cohnii and a commercial polyunsaturated fatty acid concentrate derived from fish oils (0.16 +/- 0.01%), both components of human dietary supplements. It was not found in whole bovine retina, cultured Y79 human retinoblastoma cells, or neonate baboon cerebral cortex. The long chain polyunsaturates present in the C. cohnii oil suggest a possible route for 28:8n-3 biosynthesis similar to that for biosynthesis of 22:6n-3.

Acetonitrile chemical ionization tandem mass spectrometry to locate double bonds in polyunsaturated fatty acid methyl esters.

A rapid method is presented for determining the location of double bonds in polyunsaturated fatty acid methyl esters (FAME) using an ion-trap mass spectrometer. The mass spectrum of the chemical ionization reagent acetonitrile in an ion trap includes a m/z 54 ion, identified previously as 1-methyleneimino-1-ethenylium ion. We show that it reacts with double bonds of polyunsaturated FAME to yield a series of covalent product ions all appearing at (M + 54)+. Collisional dissociation of these ions yields diagnostic fragments, permitting unambiguous localization of double bonds. For methylene-interrupted and conjugated FAME, one of these fragments results from loss of the hydrocarbon end of the chain, while the other involves loss of the methyl ester. Major diagnostic-fragment ions for monoene and diene FAME occur as a result of cleavage adjacent to either allylic sites or double bonds in the original analyte and appear at one mass unit above the mass expected for homolytic cleavage. Fragmentation of polyene FAME yields major diagnostic ions resulting from cleavage between double bonds that appear one mass unit lower. The method is shown to produce highly characteristic spectra for FAME with 1 to 6 double bonds. Identification of double-bond position in highly unsaturated fatty acids is demonstrated in a mixture of unknown polyunsaturated FAME from an extract of cultured Y79 human retinoblastoma cells.

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.

Gas chromatograph injection liner for continuous analyte admission into a mass spectrometer.

An inexpensive modification to a gas chromatography injector liner is reported that facilitates continuous admission of analyte into a gas chromatograph/mass spectrometer (GC/MS) for methods development. The MS methods development liner can be made by making simple modifications to commercially available liners and fits into standard injectors in place of the normal liners without any need to break vacuum in the MS. The injector temperature and gas flow rates are adjusted to provide appropriate analyte levels in the MS, which can be admitted under conditions identical with those of real analyses, including co-admission of column bleed. The device is particularly useful for development of tandem MS methods in GC/MS/MS instruments, which are configured with the GC as the sole sample inlet.

High-precision D/H measurement from organic mixtures by gas chromatography continuous-flow isotope ratio mass spectrometry using a palladium filter.

Continuous-flow high-precision determination of D/H ratios from an organic mixture is described using gas chromatography coupled to a Pd filter system as an interface for isotope ratio mass spectrometry. A gas chromatograph and combustion and reduction furnaces are connected to a Pd filter via a postcolumn head pressure makeup gas to increase chromatographic sensitivity. This interface is evaluated using benzene as an internal standard in a mixture of ethylbenzene and cyclohexanone in hexane with analyte quantities of < 3 ng (< 300 pg of H). A calibration curve is constructed using four benzene samples over a range of -48 to 372/1000 (delta DSMOW), resulting in an average benzene D/H precision of SD < 5/1000 (delta DSMOW) and deviations of < 4/1000 from the calibration curve. Ethylbenzene and cyclohexanone of a single D enrichment are analyzed as unknowns in three sample mixtures with varying D-enriched benzene and result in precisions of SD < 5/1000. No apparent memory is observed between peaks of differently enriched analytes within the same chromatogram. All results are corrected for ion source nonlinearities characteristic of hydrogen analysis, using the internal peakwise correction algorithm, described previously. A small dependence of isotope ratio on palladium membrane temperature is demonstrated over a range of 4 degrees C; therefore, with tighter control of palladium temperature, precision can probably be improved. The data indicate that this system is useful for rapid continuous-flow IRMS analysis of D/H ratios from organics in complex mixtures characteristic of geological and biological samples.

The expression of cytosolic phospholipase A2 and prostaglandin endoperoxide synthase in ovine maternal uterine and fetal tissues during late gestation and labor.

Gene expression of cytosolic phospholipase A2 (cPLA2) and prostaglandin endoperoxide synthase (PGHS) 1 and 2 was studied in ovine maternal uterine and fetal tissues during glucocorticoid induced premature labor and spontaneous labor and compared with gestational age matched controls not in labor. Northern blot analysis demonstrated that the messenger RNA (mRNA) abundance of cPLA2 and PGHS-2, but not PGHS-1, increased significantly in the endometrium during both glucocorticoid induced premature labor and spontaneous labor. Protein levels of the two enzymes measured by Western blot analyses were also elevated in the endometrium during glucocorticoid induced premature labor. cPLA2 mRNA was detected in the myometrium, but no difference was observed between ewes in labor and their gestational age matched controls not in labor. While the level of PGHS-2 mRNA was below detection in the myometrium with the methods used, the enzyme protein for PGHS-2 in the myometrium was significantly increased in both glucocorticoid induced premature labor and spontaneous labor. Using immunocytochemical methods, PGHS-2 enzyme protein was identified in the endometrial gland epithelial cells and myometrial cells. PGHS-2 mRNA was also detected in the ovine fetal cotyledons in which no differences for PGHS-2 mRNA existed between tissues collected from ewes in labor and ewes not in labor. In contrast, there were no detectable signals for cPLA2 or PGHS-2 mRNA in either amnion or chorion. We conclude that the ovine endometrium and myometrium, but not fetal membranes, are important tissue sites of prostaglandin biosynthesis involved in the process of ovine parturition.

Distribution of arachidonic, eicosapentaenoic, docosahexaenoic and related fatty acids in ovine endometrial phospholipids in late gestation and labor.

The quantitative distribution of phospholipid (PL) fatty acids from ovine endometrial tissues taken at 105 (n = 3) and 131 and 147 (n = 5) days of gestation age (dGA) and in spontaneous labor (SL, n = 3) is reported. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), and lysophosphatidylcholine (LPC), were separated by thin layer chromatography (TLC) and analyzed for fatty acid composition by quantitative gas chromatography (GC). Saturates are found mainly in PS and PI and unsaturates predominantly in PC and PE. The major long-chain polyunsaturated fatty acids (LC-PUFA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), are found primarily in PC, PE, and PI. AA accumulates in PC, PI and PS (p < 0.05) from late gestation to term and significantly declines in PC and PS (p < 0.02) during labor, suggesting that ovine endometrium is a possible source of prostaglandin (PG) precursors. EPA decreases significantly from around 105 dGA to term and at labor in PC (p < 0.02) and in PI (p < 0.01), which may indicate the involvement of 3-series PGs in the regulation of uterine contraction. Unsaturation index (UI) and total PUFA increase from late gestation to term in PE (p < 0.05) and decrease during labor (p < 0.05). The ratios of n-6/n-3 PUFA increase in PI (p < 0.05) and in PC (p < 0.01) during labor mainly due to the decline of EPA in these PL.(ABSTRACT TRUNCATED AT 250 WORDS)

High-precision D/H measurement from hydrogen gas and water by continuous-flow isotope ratio mass spectrometry.

Two instrumental approaches are described for continuous-flow high-precision determinations of D/H ratios from hydrogen gas or via on-line reduction of water. In the first system, Ar is used as a carrier gas, with a Ni reduction furnace and a water trap to remove minor levels of unreduced water that are a potential source of memory effects. Precisions of SD < 10/1000 (delta DSMOW) over a 600/1000 range from -55 to +532/1000 are obtained for liquid water (0.4 microL). Linearity is excellent over 4 orders of magnitude of D concentration in tap water (r2 > 0.9999), although precision degrades at enrichments delta DSMOW > 5000/1000. In the second system, a heated Pd metal foil functions as a filter to admit purified hydrogen into the mass spectrometer. Hydrogen gas injections are made into flowing Ar and are directed to the Pd filter (approximately 330 degrees C) which passes hydrogen isotopes only while diverting the carrier flow to waste. Precisions of these measurements are SD < 6/1000 over the D enrichment range -213 to 340/1000, with excellent linearity (r2 > 0.9999) and accuracy (< 2/1000). Similar precision is obtained using the on-line reduction apparatus and a water trap prior to the Pd filter with injections of 0.4 microL of liquid water, with acceptable linearity (r2 > 0.999) over 3 orders of magnitude of D concentration. Neither system shows any sign of memory effects when water is analyzed. The data indicate that either one of these systems is a useful means for continuous-flow IRMS of D/H isotope ratio determinations.

High-precision gas chromatography-combustion isotope ratio mass spectrometry at low signal levels.

Precision and accuracy of gas chromatography-combustion isotope ratio mass spectrometry are investigated for sample levels down to about 5 pmol C in fatty acid methyl ester mixtures spanning 1000-fold in concentration. Precision and accuracy of isotope ratios diverge rapidly for conventional summation methods, and become unusable below 30 pmol material on column. At lower levels, mean isotope ratios were statistically different from reference values indicating bias as well as poor precision. In contrast, curve fitting, using the exponentially modified Gaussian line shape, gives improved precision for most peaks and useful results down to 3 pmol. The curve-fitting algorithm was also less sensitive to signal integration time than the summation method. These data indicate that curve fitting may be the method of choice for integration of noisy data when high-precision isotope ratios are desired.

Curve fitting for restoration of accuracy for overlapping peaks in gas chromatography/combustion isotope ratio mass spectrometry.

The effect of graded degrees of overlap on high-precision and -accuracy carbon isotope ratios determined by gas chromatography/combustion isotope ratio mass spectrometry (GCC/IRMS) is reported. Overlapping peaks of closely matched isotope ratio (difference delta 13CPDB < 1%) were analyzed by the conventional vertical drop summation algorithm and by curve fitting using the Levenberg-Marquardt algorithm. The conventional algorithm resulted in systematic bias related to degree of overlap even though precision was not noticeably affected. The exponentially modified Gaussian (EMG) and HaarhoffVanderLinde (HVL) functions were found to model GCC/IRMS peaks satisfactorily. Useful models over a wide range of overlap were obtained by applying consecutive HVL/HVL or HVL/EMG functions to overlapping peaks. Accuracy was improved in most cases and was never degraded. This study demonstrates the presence of subtle bias in isotope ratio determinations of overlapping peaks and the ability of automated curve fitting to compensate for these biases.

High sensitivity tracer detection using high-precision gas chromatography-combustion isotope ratio mass spectrometry and highly enriched [U-13C]-labeled precursors.

The use of highly enriched, uniformly labeled fatty acid ([U-13C]) with analysis by high-precision gas chromatography-combustion isotope ratio mass spectrometry (GCC-IRMS) has been evaluated as a metabolic tracer technique. 13C/12C ratios are routinely determined to precisions (SD) of less than 0.00001 (delta PDB less than 1/1000) for greater than 10 ng of fatty acid, and less than 0.001 (delta PDB less than 100/1000) for samples of 30 pg of fatty acid, the latter corresponding to a 100-fmol sample. Baseline fatty acid 13C/12C in human plasma fractions is shown to fluctuate not more than 0.000 04 (delta PDB 4/1000) over 10 h. 13C/12C enrichments greater than 0.001 (delta PDB 100/1000) are obtained in a fatty acid plasma fraction subsequent to a 10-mg dose of 42% 13C-labeled stearic acid to a 78-kg adult. Biokinetics are discerned over an 13C/12C enrichment range of less than 0.0002 (approximately 13/1000 in delta PDB units) in plasma. A means for correction of isotope ratio contamination due to carbon-containing derivatives is presented. High-precision GCC-IRMS used in concert with highly enriched tracers is shown to possess advantages versus organic GC/MS for stable isotopic tracer detection and is superior to radiotracer methods in terms of dose sizes and analysis efficiency.