Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection

We describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their aglycone structures, 2-methyl-3-(3'-3'-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone) and 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentenyl)-1,4-naphthoquinone (7C-aglycone), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C18) solid-phase extraction (SPE), and the predominantly conjugated vitamin K metabolites were hydrolyzed with methanolic HCl. The resulting carboxylic acid aglycones were quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis was by reversed-phase (C18) HPLC with a methanol-aqueous mobile phase. Metabolites were detected by amperometric, oxidative ECD of their quinol forms, which were generated by postcolumn coulometric reduction at an upstream electrode. The assay gave excellent linearity (typically, r2 > or = 0.999) and high sensitivity with an on-column detection limit of < 3.5 fmol (< 1 pg). The interassay precision was typically 10%. Metabolite recovery was compared with that of an internal standard [2-methyl-3-(7'-carboxy-heptyl)-1,4-naphthoquinone] added to urine samples just before analysis. Using this methodology, we confirmed that the 5C- and 7C-aglycones were major catabolites of both phylloquinone (vitamin K1) and menaquinones (vitamin K2) in humans. We propose that the measurement of urinary vitamin K metabolite excretion is a candidate noninvasive marker of total vitamin K status.     

Separation and identification of neisserial lipooligosaccharide oligosaccharides using high-performance anion-exchange chromatography with pulsed amperometric detection

We determined the optimal conditions for high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) of oligosaccharides (OS) released from neisserial lipooligosaccharides (LOS) by mild acid hydrolysis. We efficiently obtained detailed composition, sequence, and linkage information about high Mr LOS. We found that HPAE-PAD can discriminate isobaric (same Mr) molecules of different structure, for example, nLc4 and Gb4, distinguish alpha from beta chain extensions, and determine the number of phosphoethanolamine (PEA) substituents. HPAE-PAD provided quantitative information that could be used to compare the relative abundances of OS. We used HPAE-PAD to identify all of the known LOS alpha chain antennae. When used with antibody-binding profiles and exoglycosidase digestion results, HPAE-PAD can provide nearly complete structures rapidly.     

Dipalmitoylation of a cellular uptake-mediating apolipoprotein E-derived peptide as a promising modification for stable anchorage in liposomal drug carriers

Liposomes equipped with cellular uptake-mediating peptidic vector compounds have attracted much attention as target-specific drug delivery systems. Aside from the development of the target recognition motif itself, vector coupling to liposomes while conserving the active conformation constitutes an important element in carrier development. To elucidate the most efficient way for adsorptive peptide binding to liposomes, we synthesized and characterized two-domain peptides comprising a cationic sequence derived from the binding domain of apolipoprotein E (apoE) for the low-density lipoprotein receptor and different lipid-binding motifs, that is, an amphipathic helix, a transmembrane helix, single fatty acids or two palmitoyl chains. Peptide properties considered relevant for peptide-liposome complexes to initiate an endocytotic cellular uptake such as lipid binding, helicity, stability of anchorage, bilayer-disturbing activity, and toxicity showed that the dipalmitoyl derivative was the most suitable to associate the apoE peptide to the surface of liposomes. The peptide showed pronounced lipid affinity and was stably anchored within the lipid bilayer on a time scale of at least 30 min. The helicity of about 40% in the lipid-bound state and the location of the amphipathic helix on the liposomal surface provided the prerequisites for interaction of the complex with the cell surface-located receptor. The concentration of the dipalmitoylated peptide to permeabilize neutral lipid bilayers (lipid concentration 25 μM) was 0.06 μM and a 2 μM concentration reduced cell viability to about 80%. Efficient internalization of liposomes bearing about 180 peptide derivatives on the surface into brain capillary endothelial cells was monitored by confocal laser scanning microscopy. The concept of complexation using dipalmitoylated peptides may offer an efficient substitute to covalent vector coupling and a prospective way to optimize the capacity of liposomes as drug delivery systems also for different targets.     

Biochemical characterization of a novel cycloisomaltooligosaccharide glucanotransferase from Paenibacillus sp. 598K

Cycloisomaltooligosaccharide glucanotransferase (CITase; EC 2.4.1.248), a member of the glycoside hydrolase family 66 (GH66), catalyzes the intramolecular transglucosylation of dextran to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) of varying lengths. Eight CI-producing bacteria have been found; however, CITase from Bacillus circulans T-3040 (CITase-T3040) is the only CI-producing enzyme that has been characterized to date. In this study, we report the gene cloning, enzyme characterization, and analysis of essential Asp and Glu residues of a novel CITase from Paenibacillus sp. 598K (CITase-598K). The cit genes from T-3040 and 598K strains were expressed recombinantly, and the properties of Escherichia coli recombinant enzymes were compared. The two CITases exhibited high primary amino acid sequence identity (67%). The major product of CITase-598K was cycloisomaltoheptaose (CI-7), whereas that of CITase-T3040 was cycloisomaltooctaose (CI-8). Some of the properties of CITase-598K are more favorable for practical use compared with CITase-T3040, i.e., the thermal stability for CITase-598K (≤ 50 °C) was 10 °C higher than that for CITase-T3040 (≤ 40 °C); the k_cat/K_M value of CITase-598K was approximately two times higher (32.2 s^− 1 mM^− 1) than that of CITase-T3040 (17.8 s^− 1 mM^− 1). Isomaltotetraose was the smallest substrate for both CITases. When isomaltoheptaose or smaller substrates were used, a lag time was observed before the intramolecular transglucosylation reaction began. As substrate length increased, the lag time shortened. Catalytically important residues of CITase-598K were predicted to be Asp144, Asp269, and Glu341. These findings will serve as a basis for understanding the reaction mechanism and substrate recognition of GH66 enzymes.     

Analysis of single nucleotide polymorphisms in the promoter region of interleukin-10 by denaturing high-performance liquid chromatography.

Interleukin-10 (IL10), an anti-inflammatory cytokine, has been implicated in a variety of immune- and inflammatory-related diseases. We investigated the following SNPs: -1082, -819, -592 in the promoter region of IL10 in a normal (control) population and selected diseases: breast cancer (BrCa), systemic lupus erythematosus (SLE), and B-cell chronic lymphocytic leukemia (B-CLL) by denaturing high-performance liquid chromatography (DHPLC) and found distinct genotype and haplotype patterns. DHPLC was performed using the Transgenomic WAVE instrument, a mutational discovery tool that allows for high throughout analysis of SNPs. The principle of DHPLC is based on separation of homo- and heteroduplex formation of individual polymerase chain reaction products at specific melting temperatures and set gradients. The melting temperature selected for each SNP was based on size and sequence of the polymerase chain reaction product (for -1082, 57 degrees C; for -819, 58 degrees C; and for -592, 59.2 degrees C). Before fragment mutational analysis, all samples were denatured at 95 degrees C and slowly reannealed to allow for reassociation of different strands. Heteroduplex samples were easily distinguished from homoduplex samples. In order to identify wild type from homozygous mutant, two homoduplex polymerase chain reaction samples had to be mixed together, denatured at 95 degrees C and reannealed. The homozygous mutant, when combined with wild type, displayed a double peak on chromatogram. Once distinct chromatograms were established for each of the SNPs and the nucleotide changes confirmed by sequencing, genotype and haplotype frequencies were tabulated for the groups studied.     

Streptozotocin diabetes results in increased responsiveness of adipocyte lipolysis to glucagon

Adipocytes from streptozotocin-diabetic rats are approximately 50-times more sensitive to the lipolytic action of glucagon. This change is only perceived in the presence of a small quantity of adenosine deaminase which itself has little effect on basal lipolysis. Insulin treatment restores glucagon sensitivity to normal.     

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF PHYTOPLANKTON PIGMENTS USING A POLYMERIC REVERSED-PHASE C18 COLUMN1

A high-performance liquid chromatographic (HPLC) method is described that allows improved resolution of several chemotaxonomically significant phytoplankton pigments. The protocol, which employs two pumps and a modified Mantoura and Llewellyn (1983) solvent system, can be easily adapted for many HPLC systems currently in use. The most unique aspect of the method is the use of a polymeric C₁₈ reversed phase HPLC column (VYDAC 201TP). In comparison to the monomeric C₁₈ columns typically used in the characterization of phytoplankton pigments, polymeric C₁₈ columns offer superior selectivity for structurally similar compounds. The protocol was evaluated for the ability to resolve most of the phytoplankton pigments of diagnostic importance using algal cultures from nine classes. Pigment pairs that were resolved by the method include a) lutein and zeaxanthin, b) neoxanthin and 19′-hexanoyloxyfucoxanthin, and c) α-carotene and β-carotene, and partial resolution of chlorophyll c₁ and chlorophyll c₂.     

Natural products as modulators of the nuclear receptors and metabolic sensors LXR,FXR and RXR

Nuclear receptors (NRs) represent attractive targets for the treatment of metabolic syndrome-related diseases. In addition, natural products are an interesting pool of potential ligands since they have been refined under evolutionary pressure to interact with proteins or other biological targets.This review aims to briefly summarize current basic knowledge regarding the liver X (LXR) and farnesoid X receptors (FXR) that form permissive heterodimers with retinoid X receptors (RXR). Natural product-based ligands for these receptors are summarized and the potential of LXR, FXR and RXR as targets in precision medicine is discussed.     

Transformation of leukotriene A4 methyl ester to leukotriene C4 monomethyl ester by cytosolic rat glutathione transferases

Six major basic cytosolic glutathione transferases from rat liver catalyzed the conversion of leukotriene A4 methyl ester to the corresponding leukotriene C4 monomethyl ester. Glutathione transferase 4-4, the most active among these enzymes, had a Vmax of 615 nmol X min-1 X mg protein-1 at 30 degrees C in the presence of 5 mM glutathione. It was followed in efficiency by transferase 3-4 which had a Vmax of 160 nmol X min-1 X mg-1 under the same conditions. Transferases 1-1, 1-2, 2-2 and 3-3 had at least 30 times lower Vmax values than transferase 4-4.