Rinat Ran-Ressler

Branched Chain Fatty Acids Are Constituents of the Normal Healthy Newborn Gastrointestinal Tract

Vernix suspended in amniotic fluid is normally swallowed by the late term fetus. We hypothesized that branched chain fatty acids (BCFA), long known to be major vernix components, would be found in meconium and that the profiles would differ systematically. Vernix and meconium were collected from term newborns and analyzed. BCFA-containing lipids constituted about 12% of vernix dry weight, and were predominantly saturated, and had 11–26 carbons per BCFA. In contrast, meconium BCFA had 16–26 carbons, and were about 1% of dry weight. Meconium BCFA were mostly in the iso-configuration, whereas vernix BCFA contained dimethyl and middle chain branching, and five anteiso-BCFA. The mass of BCFA entering the fetal gut as swallowed vernix particles is estimated to be 180 mg in the last month of gestation whereas the total mass of BCFA found in meconium is estimated to be 16 mg, thus most BCFA disappear from the fetal gut. The BCFA profiles of vernix and meconium show that BCFA are major components of normal healthy term newborn gastrointestinal tract. BCFA are candidates for agents that play a role in gut colonization and should be considered a nutritional component for the fetus/newborn.

Dietary zinc deficiency affects blood linoleic acid: dihomo-γ-linolenic acid (LA:DGLA) ratio; a sensitive physiological marker of zinc status in vivo (Gallus gallus).

Zinc is a vital micronutrient used for over 300 enzymatic reactions and multiple biochemical and structural processes in the body. To date, sensitive and specific biological markers of zinc status are still needed. The aim of this study was to evaluate Gallus gallus as an in vivo model in the context of assessing the sensitivity of a previously unexplored potential zinc biomarker, the erythrocyte linoleic acid: dihomo-γ-linolenic acid (LA:DGLA) ratio. Diets identical in composition were formulated and two groups of birds (n = 12) were randomly separated upon hatching into two diets, Zn(+) (zinc adequate control, 42.3 μg/g zinc), and Zn(−) (zinc deficient, 2.5 μg/g zinc). Dietary zinc intake, body weight, serum zinc, and the erythrocyte fatty acid profile were measured weekly. At the conclusion of the study, tissues were collected for gene expression analysis. Body weight, feed consumption, zinc intake, and serum zinc were higher in the Zn(+) control versus Zn(−) group (p < 0.05). Hepatic TNF-α, IL-1β, and IL-6 gene expression were higher in the Zn(+) control group (p < 0.05), and hepatic Δ6 desaturase was significantly higher in the Zn(+) group (p < 0.001). The LA:DGLA ratio was significantly elevated in the Zn(−) group compared to the Zn(+) group (22.6 ± 0.5 and 18.5 ± 0.5, % w/w, respectively, p < 0.001). This study suggests erythrocyte LA:DGLA is able to differentiate zinc status between zinc adequate and zinc deficient birds, and may be a sensitive biomarker to assess dietary zinc manipulation.

Structural characterization of saturated branched chain fatty acid methyl esters by collisional dissociation of molecular ions generated by electron ionization

Saturated branched chain fatty acids (BCFA) are present as complex mixtures in numerous biological samples. The traditional method for structure elucidation, electron ionization (EI) mass spectrometry, sometimes does not unambiguously enable assignment of branching in isomeric BCFA. Zirrolli and Murphy (Zirrolli , J. A. , and R. A. Murphy. 1993. Low-energy tandem mass spectrometry of the molecular ion derived from fatty acid methyl esters: a novel method for analysis of branched-chain fatty acids. J. Am. Soc. Mass Spectrom. 4: 223–229.) showed that the molecular ions of four BCFA methyl ester (BCFAME) yield highly characteristic fragments upon collisional dissociation using a triple quadrupole instrument. Here, we confirm and extend these results by analysis using a tabletop 3-D ion trap for activated molecular ion EI-MS/MS to 30 BCFAME. iso-BCFAME produces a prominent ion (30-100% of base peak) for [M-43] (M-C3H7), corresponding to the terminal isopropyl moiety in the original iso-BCFAME. Anteiso-FAME yield prominent ions (20-100% of base peak) corresponding to losses on both side of the methyl branch, [M-29] and [M-57], and tend to produce more prominent m/z 115 peaks corresponding to a cyclization product around the ester. Dimethyl and tetramethyl FAME, with branches separated by at least one methylene group, yield fragment on both sides of the sites of methyl branches that are more than 6 C away from the carboxyl carbon. EI-MS/MS yields uniquely specific ions that enable highly confident structural identification and quantification of BCFAME.

Branched-chain fatty acid composition of human milk and the impact of maternal diet: the Global Exploration of Human Milk (GEHM) Study

BACKGROUND An understudied component of the diet, branched-chain fatty acids (BCFAs) are distinctive saturated fatty acids that may have an important influence on health. Human-milk fatty acid composition is known to differ worldwide, but comparative data are lacking on BCFAs. OBJECTIVE We tested the hypotheses that concentrations of BCFAs in human milk differ between populations and are associated with maternal diet. DESIGN We surveyed the BCFA composition of samples collected as part of a standardized, prospective study of human-milk composition. Mothers were enrolled from 3 urban populations with differing diets: Cincinnati, Ohio; Shanghai, China; and Mexico City, Mexico. Enrollment was limited to healthy mothers of term singleton infants. We undertook a cross-sectional analysis of milk from all women with samples at postpartum week 4 (n = 359; ∼120 women/site). Fatty acids were extracted from milk by using a modified Bligh-Dyer technique and analyzed by gas chromatography. Statistical analysis was performed by ANOVA and Tobit regression. For Cincinnati mothers, 24-h diet recalls were analyzed in relation to the individual BCFA concentrations measured in milk samples. RESULTS Total BCFAs in milk differed by site, with the highest concentration in Cincinnati followed by Mexico City and Shanghai (mean ± SE: 7.90 ± 0.41, 6.10 ± 0.36, and 4.27 ± 0.25 mg/100 mL, respectively; P < 0.001). Site differences persisted after delivery mode, maternal age, and body mass index were controlled for. The individual concentrations of iso-14:0, iso-16:0, iso-18:0, anteiso-15:0, and anteiso-17:0 also differed between sites. Milk concentrations of iso-14:0 and anteiso-15:0 were associated with maternal intake of dairy; iso-16:0 was associated with maternal intakes of dairy and beef. CONCLUSIONS BCFA concentrations in milk at 4 wk postpartum differed between mothers from Cincinnati, Shanghai, and Mexico City. Variations in human-milk BCFAs are influenced by diet. The impact of BCFAs on infant health warrants investigation.

Sea Lions Develop Human-like Vernix Caseosa Delivering Branched Fats and Squalene to the GI Tract

Vernix caseosa, the white waxy coating found on newborn human skin, is thought to be a uniquely human substance. Its signature characteristic is exceptional richness in saturated branched chain fatty acids (BCFA) and squalene. Vernix particles sloughed from the skin suspended in amniotic fluid are swallowed by the human fetus, depositing BCFA/squalene throughout the gastrointestinal (GI) tract, thereby establishing a unique microbial niche that influences development of nascent microbiota. Here we show that late-term California sea lion (Zalophus californianus) fetuses have true vernix caseosa, delivering BCFA and squalene to the fetal GI tract thereby recapitulating the human fetal gut microbial niche. These are the first data demonstrating the production of true vernix caseosa in a species other than Homo sapiens. Its presence in a marine mammal supports the hypothesis of an aquatic habituation period in the evolution of modern humans.

A novel FADS2 isoform identified in human milk fat globule suppresses FADS2 mediated Δ6-desaturation of omega-3 fatty acids

INTRODUCTION The only known non-pharmacological means to alter long chain polyunsaturated fatty acid (LCPUFA) abundance in mammalian tissue is by altering substrate fatty acid ratios. Alternative mRNA splicing is increasingly recognized as a modulator of protein structure and function. Here we report identification of a novel alternative transcript (AT) of fatty acid desaturase 2 (FADS2) that inhibits production of omega-3 but not omega-6 LCPUFA, discovered during study of ATs in human milk fat globules (MFG). METHODS Human breastmilk collected from a single donor was used to isolate MFG. An mRNA-sequencing library was constructed from the total RNA isolated from the MFG. The constructed library was sequenced using an Illumina HiSeq instrument operating in high output mode. Expression levels of evolutionary conserved FADSAT were measured using cDNA from MFG by semi-quantitative RT-PCR assay. RESULTS RNA sequencing revealed >15,000 transcripts, including moderate expression of the FADS2 classical transcript (CS). A novel FADS2 alternative transcript (FADS2AT2) with 386 amino acids was discovered. When FADS2AT2 was transiently transfected into MCF7 cells stably expressing FADS2, delta-6 desaturation (D6D) of alpha-linolenic acid 18:3n-3 → 18:4n-3 was suppressed as were downstream products 20:4n-3 and 20:5n-3. In contrast, no significant effect on D6D of linoleic acid 18:2n-6 → 18:3n-6 or downstream products was observed. FADS2, FADS2AT1 and 5 out of 8 known FADS3AT were expressed in MFG. FADS1, FADS3AT3, and FADS3AT5 are undetectable. CONCLUSION The novel, noncatalytic FADS2AT2 regulates FADS2CS-mediated Δ6-desaturation of omega-3 but not omega-6 PUFA biosynthesis. This spliced isoform mediated interaction is the first molecular mechanism by which desaturation of one PUFA family but not the other is modulated.