Inadequate Niacin Intake Disrupts Growth and Retinol Homeostasis Resulting in Higher Liver and Lower Serum Retinol Concentrations in Male Rats.

BACKGROUND: Niacin-derived nicotinamide adenine dinucleotide is an essential cofactor for many dehydrogenase enzymes involved in vitamin A (VA) metabolism. Several countries with high prevalence of VA deficiency rely on maize, a poor source of available niacin, as a dietary staple. OBJECTIVES: This study evaluated the interaction of dietary niacin on VA homeostasis using male Sprague-Dawley rats, aged 21 d (baseline body weight 88.3 ± 6.6 g). METHODS: After 1 wk of acclimation, baseline samples were collected (n = 4). Remaining rats (n = 54) were split into 9 groups to receive low tryptophan, VA-deficient feed with 3 different amounts of niacin (0, 15, or 30 mg/kg) and 3 different oral VA doses (50, 350, or 3500 nmol/d) in a 3 × 3 design. After 4 wk, the study was terminated. Serum, livers, and small intestine were analyzed for retinoids using high-performance liquid chromatography. Niacin and metabolites were evaluated with nuclear magnetic resonance. Plasma pyridoxal-P (PLP) was measured with high-performance liquid chromatography. RESULTS: Niacin intake correlated with serum retinol concentrations (r = 0.853, P < 0.001). For rats receiving the highest VA dose, liver retinol concentrations were lower in the 30-mg/kg niacin group (5.39 ± 0.27 µmol/g) than those in the 0-mg/kg and 15-mg/kg groups (9.18 ± 0.62 and 8.75 ± 0.07 µmol/g, respectively; P ≤ 0.05 for both). This phenomenon also occurred in the lower VA doses (P ≤ 0.05 for all). Growth and tissue weight at endline were associated with niacin intake (P ≤ 0.001 for all). Plasma PLP correlated with estimated niacin intake (r = 0.814, P < 0.001). CONCLUSIONS: Optimal niacin intake is associated with lower liver VA and higher serum retinol and plasma PLP concentrations. The extent to which vitamin B intake affects VA homeostasis requires further investigation to determine if the effects are maintained in humans.

Stable Picodisc Assemblies from Saposin Proteins and Branched Detergents.

Methods for maintaining membrane proteins in their native state after removal from the lipid bilayer are essential for the study of this important class of biomacromolecules. Common solubilization strategies range from the use of detergents to more complex systems that involve a polypeptide working in concert with lipids or detergents, such as nanodiscs, picodiscs, and peptidiscs, in which an engineered protein or synthetic peptide surrounds the membrane protein along with a lipid sheath. Picodiscs employ the protein saposin A, which naturally functions to facilitate lipid degradation in the lysozome. Saposin A-amphiphile complexes therefore tend to be most stable at acidic pH, which is not optimal for most membrane protein applications. In search of new picodisc assemblies, we have explored pairings of saposin A or other saposin proteins with a range of detergents, and we have identified a number of combinations that spontaneously co-assemble at neutral pH. The resulting picodiscs are stable for weeks and have been characterized by size-exclusion chromatography, native mass spectrometry, and small angle X-ray scattering. The new assemblies are formed by double-tail detergents rather than more traditional single-tail detergents; the double-tail detergents can be seen as structurally intermediate between single-tail detergents and common lipids. In addition to saposin A, an engineered variant of saposin B (designated saposin BW) forms picodisc assemblies. These findings provide a framework for future efforts to solubilize membrane proteins with multiple picodisc systems that were previously unknown.

Metabolic Changes in Synaptosomes in an Animal Model of Schizophrenia Revealed by 1H and 1H,13C NMR Spectroscopy.

Synaptosomes are isolated nerve terminals that contain synaptic components, including neurotransmitters, metabolites, adhesion/fusion proteins, and nerve terminal receptors. The essential role of synaptosomes in neurotransmission has stimulated keen interest in understanding both their proteomic and metabolic composition. Mass spectrometric (MS) quantification of synaptosomes has illuminated their proteomic composition, but the determination of the metabolic composition by MS has been met with limited success. In this study, we report a proof-of-concept application of one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy for analyzing the metabolic composition of synaptosomes. We utilize this approach to compare the metabolic composition synaptosomes from a wild-type rat with that from a newly generated genetic rat model (Disc1 svΔ2), which qualitatively recapitulates clinically observed early DISC1 truncations associated with schizophrenia. This study demonstrates the feasibility of using NMR spectroscopy to identify and quantify metabolites within synaptosomal fractions.

NMR-Based Identification of Metabolites in Polar and Non-Polar Extracts of Avian Liver.

Metabolites present in liver provide important clues regarding the physiological state of an organism. The aim of this work was to evaluate a protocol for high-throughput NMR-based analysis of polar and non-polar metabolites from a small quantity of liver tissue. We extracted the tissue with a methanol/chloroform/water mixture and isolated the polar metabolites from the methanol/water layer and the non-polar metabolites from the chloroform layer. Following drying, we re-solubilized the fractions for analysis with a 600 MHz NMR spectrometer equipped with a 1.7 mm cryogenic probe. In order to evaluate the feasibility of this protocol for metabolomics studies, we analyzed the metabolic profile of livers from house sparrow (Passer domesticus) nestlings raised on two different diets: livers from 10 nestlings raised on a high protein diet (HP) for 4 d and livers from 12 nestlings raised on the HP diet for 3 d and then switched to a high carbohydrate diet (HC) for 1 d. The protocol enabled the detection of 52 polar and nine non-polar metabolites in ¹H NMR spectra of the extracts. We analyzed the lipophilic metabolites by one-way ANOVA to assess statistically significant concentration differences between the two groups. The results of our studies demonstrate that the protocol described here can be exploited for high-throughput screening of small quantities of liver tissue (approx. 100 mg wet mass) obtainable from small animals.