Vitamin C depletion increases superoxide generation in brains of SMP30/GNL knockout mice

Vitamin C (VC) has a strong antioxidant function evident as its ability to scavenge superoxide radicals in vitro. We verified that this property actually exists in vivo by using a real-time imaging system in which Lucigenin is the chemiluminescent probe for detecting superoxide in senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize VC in vivo. SMP30/GNL KO mice were given 1.5 g/L VC [VC(+)] for 2, 4, or 8 weeks or denied VC [VC(−)]. At 4 and 8 weeks, VC levels in brains from VC(−) KO mice were <6% of that in VC(+) KO mice. Accordingly, superoxide-dependent chemiluminescence levels determined by ischemia-reperfusion at the 4- and 8 weeks test intervals were 3.0-fold and 2.1-fold higher, respectively, in VC(−) KO mice than in VC(+) KO mice. However, total superoxide dismutase activity and protein levels were not altered. Thus, VC depletion specifically increased superoxide generation in a model of the living brain.     

Serum LDL- and HDL-cholesterol determined by ultracentrifugation and HPLC

Simple and precise methods for LDL-cholesterol (LDL-C) and HDL-cholesterol (HDL-C) measurements are essential for assessment of cardiovascular disease (CVD) risks and for lipid and lipoprotein studies. We report here an ultracentrifugation (UC) and HPLC method that requires substantially less specimen volume and provides the necessary reliability and throughput required by large-volume, high-quality research and clinical studies. 2-Mercaptoethanol (ME) was used to dissociate serum lipoprotein [a] (Lp[a]) into apolipoprotein [a] and Lp[a] remnant (Lp[a-]) and eliminated the contamination of Lp[a] in HDL separated by UC. Serum aliquots were centrifuged at a density of 1.006 kg/l for the separation of HDL plus LDL, and in the presence of ME at a density of 1.063 kg/l for the separation of HDL. Cholesterol concentrations of the bottom fractions were analyzed by HPLC. LDL-C and HDL-C determined using this method were equivalent to those with β-quantification and the designated comparison method of the Centers for Disease Control. The total coefficient of variations for LDL-C and HDL-C were 0.65-1.12% and 0.96-2.07%, respectively. This method requires a small amount of specimen and is easy to operate. This method may be used in research or in clinical laboratories where precise and specific lipoprotein cholesterol analysis is needed.     

Quality Assessment of Bee Pollen-Honey Mixtures Using Thin-Layer Chromatography in Combination with Chemometrics

The aim of this study is to develop a rapid, effect-directed screening method for quality assessment of bee pollen-honey mixtures. The comparative antioxidant potential and phenolic content of honey, bee pollen, and the bee pollen-honey mixtures, was performed using spectrophotometry. The total phenolic content and antioxidative activity of bee pollen-honey mixtures with 20 % bee pollen share were in the range 3.03–3.11 mg GAE/g, and 6.02–6.96 mmol TE/kg, respectively, while mixtures with 30 % bee pollen share contained 3.92–4.18 mg GAE/g, and 9.69–10.11 mmol TE/kg. Chromatographic fingerprint of bee pollen-honey mixtures was performed by high-performance thin-layer chromatography with conditions developed by authors and reported for the first time. Fingerprint analysis hyphenated with chemometrics enabled authenticity assessments of honey in mixtures. Results indicate that bee pollen-honey mixtures represent a food with highly, both, nutritious characteristics and health-promoting effect.     

Quantification of 3-ketodihydrosphingosine using HPLC-ESI-MS/MS to study SPT activity in yeast Saccharomyces cerevisiae

Serine palmitoyltransferase (SPT) catalyzes the rate-limiting step of condensation of L-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine (3KDS). Here, we report a HPLC-ESI-MS/MS method to directly quantify 3KDS generated by SPT. With this technique, we were able to detect 3KDS at a level comparable to that of dihydrosphingosine in yeast Saccharomyces cerevisiae. An in vitro SPT assay measuring the incorporation of deuterated serine into deuterated 3KDS was developed. The results show that SPT kinetics in response to palmitoyl-CoA fit into an allosteric sigmoidal model, suggesting the existence of more than one palmitoyl-CoA binding site on yeast SPT and positive cooperativity between them. Myriocin inhibition of yeast SPT activity was also investigated and we report here, for the first time, an estimated myriocin K_i for yeast SPT of approximately 10 nM. Lastly, we investigated the fate of serine α-proton during SPT reaction. We provide additional evidence to support the proposed mechanism of SPT catalytic activity in regard to proton exchange between the intermediate NH₃⁺ base formed on the active Lys residue with surrounding water. These findings establish the current method as a powerful tool with significant resolution and quantitative power to study SPT activity.     

The N-terminal region of the starch-branching enzyme from Phaseolus vulgaris L. is essential for optimal catalysis and structural stability

Starch-branching enzymes (SBEs) play a pivotal role in determining the fine structure of starch by catalyzing the syntheses of alpha-1,6-branch points. They are the members of the alpha-amylase family and have four conserved regions in a central (beta/alpha)8 barrel, including the catalytic sites. Although the role of the catalytic barrel domain of an SBE is known, that of its N- and C-terminal regions remain unclear. We have previously shown that the C-terminal regions of the two SBE isozymes (designated as PvSBE1 and PvSBE2) from kidney bean (Phaseolus vulgaris L.) have different roles in branching enzyme activity. To understand the contribution of the N-terminal region to catalysis, six chimeric enzymes were constructed between PvSBE1 and PvSBE2. Only one enzyme (1Na/2Nb)-II, in which a portion of the N-terminal region of PvSBE2 was substituted by the corresponding region of PvSBE1, retained 6% of the PvSBE2 activity. The N-terminal truncated form (DeltaN46-PvSBE2), lacking 46 N-terminal residues of PvSBE2, lost enzyme activity and stability to proteolysis. To investigate the possible function of this region, three residues (Asp-15, His-24, and Arg-28) among these 46 residues were subjected to site-directed mutagenesis. The purified mutant enzymes showed nearly the same K(m) values as PvSBE2 but had lower V(max) values and heat stabilities than PvSBE2. These results suggest that the N-terminal region of the kidney bean SBE is essential for maximum enzyme activity and thermostability.     

Human gliosarcoma-associated ganglioside composition is complex and distinctive as evidenced by high-performance mass spectrometric determination and structural characterization

Gangliosides (GGs), involved in malignant alteration and tumor progression/invasiveness, are considered as tumor biomarkers or therapeutic targets. Here, we describe the first systematic GG composition characterization in human gliosarcoma versus normal brain tissue using our recently developed mass spectrometry (MS) methods, based on nano-electrospray (nano-ESI), Fourier-transform ion cyclotron resonance (FT-ICR), and chip nano-ESI quadrupole time-of-flight (QTOF), complemented by thin-layer chromatographic (TLC) analysis and quantification. Combined MS enabled detection and structural assignment of 73 distinct GG species: many more than reported so far for investigated gliomas. Apart from the 7.4-times lower total GG content, gliosarcoma contained all major brain-associated species, however, in very altered proportions, exhibiting a highly distinctive pattern: GD3 (48.9%)>GD1a/nLD1>GD2/GT3>GM3>GT1b>GM2>GM1a/GM1b/nLM1>LM1>GD1b>GQ1b. MS also revealed abundant O-Ac-GD3; its sequencing provided structural evidence to postulate a novel O-Ac-GD3 isomer O-acetylated at the inner Neu5Ac-residue, previously not structurally confirmed. The high sensitivity and mass accuracy permitted the assignment of unusual minor species: GM4, Hex-HexNAc-nLM1, Gal-GD1, Fuc-GT1, GalNAc-GT1, O-Ac-GM3, di- O-Ac-GD3O-Ac-GD3, and O-Ac-GT3, not previously reported as glioma-associated. The gliosarcoma-expressed GA2 might represent a marker distinguishing astrocytic from oligodendroglial tumors. This is, to our knowledge, so far the most complete GG composition characterization of certain glioma, which demonstrates that our MS-based approach could provide essential structural information relevant to glycosphingolipid role(s) in brain tumor biology, differential diagnosis/prognosis and novel treatment concepts.     

Quantification of lipid alkyl radicals trapped with nitroxyl radical via HPLC with postcolumn thermal decomposition

Lipid alkyl radicals generated from polyunsaturated fatty acids via chemical or enzymatic H-abstraction have been a pathologically important target to quantify. In the present study, we established a novel method for the quantification of lipid alkyl radicals via nitroxyl radical spin-trapping. These labile lipid alkyl radicals were converted into nitroxyl radical-lipid alkyl radical adducts using 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (CmdeltaP) (a partition coefficient between octanol and water is approximately 3) as a spin-trapping agent. The resulting CmdeltaP-lipid alkyl radical adducts were determined by HPLC with postcolumn online thermal decomposition, in which the adducts were degraded into nitroxyl radicals by heating at 100 degrees C for 2 min. The resulting nitroxyl radicals were selectively and sensitively detected by electrochemical detection. With the present method, we, for the first time, determined the lipid alkyl radicals generated from linoleic acid, linolenic acid, and arachidonic acid via soybean lipoxygenase-1 or the radical initiator 2,2'-azobis(2,4-dimethyl-valeronitrile).     

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.