Effect of fasting status and other pre-analytical variables on quantitation of long-chain fatty acids in red blood cells.

Long-chain fatty acids (LCFAs), including omega-3 and omega-6 fatty acids, play essential roles in health maintenance and outcomes. Insufficient intake or the inability to absorb LCFAs from the diet can cause a number of health problems. Evaluation of fatty acid profiles in plasma, serum or red blood cells (RBCs) is routinely used to monitor patients at risk of developing deficiency. Quantitation of LCFAs in RBCs offers advantages over serum/plasma due to low intra-individual variability. Fatty acid composition in RBCs also reflects long-term dietary intake, providing additional information about the patient's nutritional status. However, the literature does not currently address the impact of pre-analytical factors (conditions of RBC collection, sample handling and short-term storage) on LCFA measurements. This study evaluated the effect of several anticoagulants, interferents, different storage conditions and fasting status on quantitation of the twenty-one most abundant LCFAs in RBCs by gas chromatography negative chemical ionization-mass spectrometry (GCNCI-MS). LCFA results were assessed quantitatively (nmol/mL) or as a percent of total. Most common tube types (containing citrate, sodium heparin or EDTA) were all appropriate for blood collection. Whole blood and lysed RBCs were stable at least 24 h at room temperature and up to 7 days refrigerated. Lysed RBCs were also stable for up to three freeze/thaw cycles. The presence of icterus or lipemia did not affect results. LCFAs concentrations in RBCs did not change ~4 h after high-fat intake when the lipid concentration in circulation reaches a peak, while plasma levels of most fatty acids increased up to 40% in response. In summary, RBCs are a reliable sample type for LCFA quantitation in the clinical laboratory. In contrast to plasma or serum, RBCs isolated from non-fasting, hemolyzed or lipemic whole blood specimens are all acceptable for testing. Therefore, RBCs might be a preferable sample type for evaluation of nutritional status of young pediatric patients and in patients with conditions associated with hemolytic anemia or hyperlipidemia.

Intra-individual variability of long-chain fatty acids (C12-C24) in plasma and red blood cells.

Long-chain fatty acids (LCFA) play key roles in mammalian cells as sources of energy, structural components and signaling molecules. Given their importance in numerous physiological processes, the roles of LCFAs in health and disease have been extensively investigated. In the majority of studies, correlations are established using a single measurement in plasma or red blood cells (RBCs). Although a few studies have reported on reproducibility of individual fatty acid measurements, the comprehensive analysis of intra-individual LCFA variability has not been performed. Therefore, our goal was to determine intra-individual variability for the 22 most abundant LCFAs in both plasma and RBC samples collected from healthy individuals on a regular diet after overnight fasting. The measurements of LCFAs in RBCs were consistent throughout the course of study reflecting long-term nutritional status. In contrast, the results in plasma showed considerable LCFA intra-individual variability, even between fatty acids of the same type. Plasma intra-individual variability for omega-3 alpha-linolenic and eicosapentaenoic acids in some participants were >40% whereas the variability of docosahexaenoic acid was consistently <12.8%. Omega-6 linoleic and arachidonic acids also showed low variability in plasma. The results suggest that some LCFAs have less variability and would be more reliable as biomarkers. Reliability of biomarkers can have a profound impact on the research outcomes. Intra-individual variability of LCFAs should be taken into consideration in designing, conducting and interpreting results of clinical studies.

The polycomb complex protein mes-2/E(z) promotes the transition from developmental plasticity to differentiation in C. elegans embryos.

We have used expression profiling and in vivo imaging to characterize Caenorhabditis elegans embryos as they transit from a developmentally plastic state to the onset of differentiation. Normally, this transition is accompanied by activation of developmental regulators and differentiation genes, downregulation of early-expressed genes, and large-scale reorganization of chromatin. We find that loss of plasticity and differentiation onset depends on the Polycomb complex protein mes-2/E(Z). mes-2 mutants display prolonged developmental plasticity in response to heterologous developmental regulators. Early-expressed genes remain active, differentiation genes fail to reach wild-type levels, and chromatin retains a decompacted morphology in mes-2 mutants. By contrast, loss of the developmental regulators pha-4/FoxA or end-1/GATA does not prolong plasticity. This study establishes a model by which to analyze developmental plasticity within an intact embryo. mes-2 orchestrates large-scale changes in chromatin organization and gene expression to promote the timely loss of developmental plasticity. Our findings indicate that loss of plasticity can be uncoupled from cell fate specification.

Local cholinergic suppression of pacemaker activity in the rabbit sinoatrial node.

The effects of transmural vagal stimulation and acetylcholine (ACh) superfusion on primary and latent pacemaker cells of the rabbit sinoatrial node were studied by using microelectrodes. Both ACh and vagal stimulation lengthened atrial cycle length by 40-60% as compared with control. In the cells from the primary pacemaker area, both ACh superfusion and vagal stimulation suppressed action potential (AP) amplitude and then induced inexcitability. In contrast, cells from subsidiary pacemaker area as well as atrium remained excitable. These effects were completely reversible and also were abolished by atropine, 10(-7) M. Cholinergically induced suppression of AP amplitude is predictable based on the maximal rate of AP upstroke (dV/dt). The probability of amplitude suppression was the highest among pacemaker cells (dV/dt, <3 V/s), in which ACh suppressed amplitude in 27 (93%) of 29 cells, and vagal stimulation did so in 38 (81%) of 47 cells. With increasing upstroke velocity, the probability of amplitude suppression decreased. Inexcitability did not occur in cells whose dV/dt was >15 V/s. The suppression of AP amplitude by ACh occurred in a concentration-dependent manner: the concentration inducing suppression of amplitude in 50% of pacemaker cells was approximately 10 microM. These results indicate that cholinergic effects on typical pacemaker and subsidiary pacemaker cells are different: whereas subsidiary pacemaker cells remain excitable, typical pacemaker cells become quiescent. We hypothesize that quiescent cells create quiescent regions in the center of the sinoatrial node that might functionally be an obstacle for reentrant tachycardias.