Sep-Pak C18 Cartridge Research Articles

Facile and rapid one-step radiosynthesis of [18F]BAY94-9172 with a new precursor

[(18)F]BAY94-9172 (Florbetaben) (Compound 8) is a positron emission tomography (PET) tracer that is currently in Phase III study for in vivo mapping of fibrillar amyloid β as a pathological hallmark for Alzheimer's disease. This work reports new methods for the synthesis of [(19)F]BAY94-9172 and its two different precursors and radiosynthesis of [(18)F]BAY94-9172 with the two precursors by purification using Sep-Pak cartridge. The reference standard [(19)F]BAY94-9172 and the new precursor (Compound 9) were obtained from the reactions of (E)-4-methylamino-4'-hydroxystilbene (Compound 1) with methanesulfonic acid 2-[2-(2-fluoro-ethoxy)-ethoxy]-ethyl ester (Compound 11) and methanesulfonic acid 2-[2-(2-methanesulfonyloxy-ethoxy)-ethoxy]-ethyl ester (Compound 13), respectively. The reported precursor (Compound 6) is an N-BOC-protected mesylate compound, which was obtained from Compound 9. The one-step radiosynthesis of [(18)F]BAY94-9172 was carried out in the modified PET-MF-2V-IT-1 synthesizer by [(18)F]fluorination of the new precursor (Compound 9) and purification with plus C18 Sep-Pak cartridges and was compared with two-step one-pot radiosynthesis using the reported precursor (Compound 6) and Sep-Pak cartridge purification. For one-step radiosynthesis, the uncorrected radiochemical yield of [(18)F]BAY94-9172 was 23 ± 3% (n = 5, based on [(18)F]fluoride) within 30 min and the radiochemical purity was greater than 95%. For two-step one-pot radiosynthesis, the uncorrected radiochemical yield of [(18)F]BAY94-9172 was 17 ± 2% in 45 min (n = 4, based on [(18)F]fluoride) with the radiochemical purity being above 95% after the Sep-Pak cartridge purification. [(19)F]BAY94-9172 and the two precursors were synthesized by a short synthetic route. Compared with HPLC purification, the use of Sep-Pak purification of [(18)F]BAY94-9172 reduced the total radiosynthesis time. The one-step radiosynthesis of [(18)F]BAY94-9172 is convenient and can easily be applied to the commercial PET tracer synthesizer for automated synthesis.

Axonal Transport Proteomics Reveals Mobilization of Translation Machinery to the Lesion Site in Injured Sciatic Nerve

Investigations of the molecular mechanisms underlying responses to nerve injury have highlighted the importance of axonal transport systems. To obtain a comprehensive view of the protein ensembles associated with axonal transport in injured axons, we analyzed the protein compositions of axoplasm concentrated at ligatures following crush injury of rat sciatic nerve. LC-MS/MS analyses of iTRAQ-labeled peptides from axoplasm distal and proximal to the ligation sites revealed protein ensembles transported in both anterograde and retrograde directions. Variability of replicates did not allow straightforward assignment of proteins to functional transport categories; hence, we performed principal component analysis and factor analysis with subsequent clustering to determine the most prominent injury-related transported proteins. This strategy circumvented experimental variability and allowed the extraction of biologically meaningful information from the quantitative neuroproteomics experiments. 299 proteins were highlighted by principal component analysis and factor analysis, 145 of which correlate with retrograde and 154 of which correlate with anterograde transport after injury. The analyses reveal extensive changes in both anterograde and retrograde transport proteomes in injured peripheral axons and emphasize the importance of RNA binding and translational machineries in the axonal response to injury.

Comparative glycomic profiling in esophageal adenocarcinoma

Aberrant glycosylation has been implicated in various types of cancers. Cancerous cells with altered glycosylation of their surface proteins shed such proteins into the circulating fluids. Glycomic profiling of such fluids shows the altered glycosylation. We performed glycomic profiling of serum from patients with no known disease, Barrett's without dysplasia, with high-grade dysplasia, and with esophageal adenocarcinoma in an attempt to delineate distinct differences in glycosylation among these groups. Serum samples from patients with Barrett's metaplasia (N = 5), high-grade dysplasia (N = 11), and esophageal adenocarcinoma (N = 50) were collected; samples from 18 healthy volunteers were used as control. Serum N-glycans were enzymatically released and then applied to both C18 Sep-Pak (Waters, Milford, MA) cartridges and activated charcoal cartridges. N-glycans were permethylated and then spotted directly onto a matrix-assisted laser desorption ionization plate. Mass spectra were acquired using the Applied Biosystems 4800 MALDI TOF/TOF Analyzer (Applied Biosystems Inc, Framingham, Mass). The obtained matrix-assisted laser desorption ionization-mass spectrometry data were processed using DataExplorer files (Applied Biosystems Inc) listing m/z values and intensities. The intensities of 98 glycans were significantly different among the 3 groups; 26 of these corresponded to known glycan structures. Pairwise comparisons showed that 8 glycans were significantly different in all 3 pairwise comparisons. We demonstrated that comparative glycomic profiling of esophageal adenocarcinoma reveals a subset of glycans that can be selected as candidate biomarkers. These markers can differentiate normal from high-grade dysplasia, normal from esophageal adenocarcinoma, and high-grade dysplasia from esophageal adenocarcinoma. Further validation will be necessary to determine the clinical utility of these glycan biomarkers.

Glucose stimulates cholesterol 7α-hydroxylase gene transcription in human hepatocytes

Bile acids play important roles in the regulation of lipid, glucose, and energy homeostasis. Recent studies suggest that glucose regulates gene transcription in the liver. The aim of this study was to investigate the potential role of glucose in regulation of bile acid synthesis in human hepatocytes. High glucose stimulated bile acid synthesis and induced mRNA expression of cholesterol 7alpha-hydroxylase (CYP7A1), the key regulatory gene in bile acid synthesis. Activation of an AMP-activated protein kinase (AMPK) decreased CYP7A1 mRNA, hepatocyte nuclear factor 4alpha (HNF4alpha) protein, and binding to CYP7A1 chromatin. Glucose increased ATP levels to inhibit AMPK and induce HNF4alpha to stimulate CYP7A1 gene transcription. Furthermore, glucose increased histone acetylation and decreased H3K9 di- and tri-methylation in the CYP7A1 chromatin. Knockdown of ATP-citrate lyase, which converts citrate to acetyl-CoA, decreased histone acetylation and attenuated glucose induction of CYP7A1 mRNA expression. These results suggest that glucose signaling also induces CYP7A1 gene transcription by epigenetic regulation of the histone acetylation status. This study uncovers a novel link between hepatic glucose metabolism and bile acid synthesis. Glucose induction of bile acid synthesis may have an important implication in metabolic control of glucose, lipid, and energy homeostasis under normal and diabetic conditions.

Flow-Injection Determination of Benzimidazole Fungicides in Natural Waters with Copper(II)-Hydrogen Peroxide Chemiluminescence

A simple, sensitive, and rapid flow-injection method is reported for the determination of benzimidazole fungicides (BFs): fuberidazole, benomyl, and carbendazim. The method is based on enhancement of the chemiluminescence (CL) signal generated during the reaction of BFs with hydrogen peroxide and copper(II) in alkaline condition in the absence of luminescent reagents. Linear calibration graphs were obtained in the concentration range of 5–2000, 25–2000, and 25–5000 ng mL−1 for fuberidazole, benomyl, and carbendazim, respectively. The relative standard deviations (n = 4) were in the range 1.2–2.6% with limits of detection (S/N = 3) 3 ng mL−1 for fuberidazole, and 15 ng mL−1 for benomyl and carbendazim. The proposed method allowed 120 injections h−1. The effect of common anions, cations, and some pesticides on the determination of BFs was also examined. The method is applied to determine BFs in spiked natural water samples using Sep-Pak C18 cartridges for solid phase extraction and preconcentration procedure. Recoveries were in the range of 94 ± 1 to 105 ± 1%. The CL mechanism is also discussed briefly.

The Endoplasmic Reticulum Chaperone Cosmc Directly Promotes in Vitro Folding of T-synthase

The T-synthase is the key beta 3-galactosyltransferase essential for biosynthesis of core 1 O-glycans (Gal beta 1-3GalNAc alpha 1-Ser/Thr) in animal cell glycoproteins. Here we describe the novel ability of an endoplasmic reticulum-localized molecular chaperone termed Cosmc to specifically interact with partly denatured T-synthase in vitro to cause partial restoration of activity. By contrast, a mutated form of Cosmc observed in patients with Tn syndrome has reduced chaperone function. The chaperone activity of Cosmc is specific, does not require ATP in vitro, and is effective toward T-synthase but not another beta-galactosyltransferase. Cosmc represents the first ER chaperone identified to be required for folding of a glycosyltransferase.

Diversity of molecular forms of plasma brain natriuretic peptide in heart failure—different proBNP-108 to BNP-32 ratios in atrial and ventricular overload

ObjectiveRecent studies have shown that plasma levels of brain natriuretic peptide (BNP)-32 and proBNP-108 are increased in heart failure (HF) and that the BNP-32 assay kit in current clinical use...

Separation and elucidation of anthocyanins in the fruit of mockstrawberry (Duchesnea indica Focke)

Anthocyanin pigments in the fruit of mockstrawberry (Duchesnea indica Focke), were extracted with 0.1% HCl in ethanol, and the crude anthocyanin extract was purified by a C18 Sep-Pak cartridge open-column chromatography. High-performance liquid chromatography (HPLC) with photodiode array detection and mass spectrometry (MS) was applied for the isolation and composition analysis of anthocyanins in the fruit of mockstrawberry and their aglycones from acid hydrolysis. Three anthocyanins were found in the fruit of mockstrawberry and they were identified as cyanidin 3-O-rutinoside (61%), peonidin 3-O-rutinoside (34%), and petunidin 3-O-rutinoside (5%), respectively, by spectroscopic methods (UV-vis and MS). The two major anthocyanins were isolated by preparative HPLC, and their chemical structures were further characterised by H1 NMR. On the basis of chromatographic data, the total anthocyanin content was 205 mg g−1 of the fresh fruit of mockstrawberry.

Molecular Form of Plasma Brain Natriuretic Peptide in Heart Failure: Different proBNP-108 to BNP-32 Ratios in Atrial and Ventricular Overload

Background: We investigated the pathophysiological significance of diversity of molecular form of plasma brain natriuretic peptide(BNP) in heart failure(HF). Methods: We measured plasma BNP-32 and proBNP-108 in normal control subjects(n=10) and patients with atrial fibrillation (AF)(n=18) and HF(n=132). We also measured BNP-32 and proBNP-108 in ventricular and atrial tissue and inpericardial fluid by specific fluorescent enzyme immunoassay following Sep-Pak C18 cartridge extraction and gel-filtration.

Some Methodic Aspects in Optimizing the Radioimmunoassay of β-Endorphin

A specific double antibody radioimmunoassay (RIA) for human beta-endorphin (beta-EP) using an antibody to synthetic beta h-endrophin was established. This antibody cross-reacted with beta-Lipotropin (beta-LPH) at 42 per cent on molar basis and allowed a usable range of 20 pg to 4 ng of beta-endorphin per ml in the assay. Comparing the efficiency of the conventional extraction procedures under various conditions using Corning glass, Vycor glass, QUSO G-32, silicic acid and controlled pore glass 75, the optimal result was obtained by Corning glass, with a recovery rate of more than 80 per cent. The most simple and rapid method with an extraction efficiency of more than 90 per cent was found to be the extraction by use of Sep-Pak C18 cartridges. The separation of beta-endorphin from beta-LPH was studied using Sephadex G-50, G-75, G-100 and Bio-Gel P-60 columns and different elution media. The use of a Sephadex G-50 column (0.9 X 55 cm) and elution with 0.1 N acetic acid-0.05 per cent HSA gave the best result. The reliability of the radioimmunoassay was shown under physiological and pathophysiological conditions.

Fractionation of pine bark extracts: selecting procyanidins

Pine bark is a forest by-product that can be profitable as a source of polyphenols by its content in procyanidins. In this work, barks of two varieties of pine (Pinus pinaster and P. radiata) were extracted with ethanol, and partially purified to obtain a fraction named FOW, that contained mainly flavan-3-ol monomers and procyanidins oligomers. The mean degree of polymerization was 2.1 for pinaster (pOW) and 2.7 for radiata (rOW). FOWs were fractionated on C18 Sep-Pak cartridges into three fractions for each variety by different organic solvents. The phenolic compositions of the fractions were analyzed using HPLC with UV diode array detection and electrospray-ionization mass spectrometry. The compounds identified were: catechin, dihydroquercetin, phenolic acids and their glucosides, procyanidins dimers, trimers and tetramers. FOWs and their fractions were characterized in antioxidant power by DPPH and β-carotene methods. In the former assay, pOW showed major effectiveness, with an antiradical specific activity of 46 units AR/mg procyanidins, whereas for the β-carotene bleaching test rOW (78 ± 0.71%) gave the best inhibition result. Despite this fact the derived fractions, presenting similar values, have advantages because the profile of their constituents is uniform for each one, and, hence, it is easier to investigate their action for potential applications in both medical or food fields

Core3 O-Glycan Synthase Suppresses Tumor Formation and Metastasis of Prostate Carcinoma PC3 and LNCaP Cells through Down-regulation of α2β1 Integrin Complex

Although there are numerous reports of carbohydrates enriched in cancer cells, very few studies have addressed the functions of carbohydrates present in normal cells that decrease in cancer cells. It has been reported that core3 O-glycans are synthesized in normal gastrointestinal cells but are down-regulated in cancer cells. To determine the roles of core3 O-glycans, we transfected PC3 and LNCaP prostate cancer cells with beta3-N-acetylglucosaminyltransferase-6 (core3 synthase) required to synthesize core3 O-glycans. Both engineered cell lines exhibited reduced migration and invasion through extracellular matrix components compared with mock-transfected cells. Moreover we found that alpha2beta1 integrin acquired core3 O-glycans in cells expressing core3 synthase with decreased maturation of beta1 integrin, leading to decreased levels of the alpha2beta1 integrin complex, decreased activation of focal adhesion kinase, and reduced lamellipodia formation. Upon inoculation into the prostate of nude mice, PC3 cells expressing core3 O-glycans produced much smaller tumors without metastasis to the surrounding lymph nodes in contrast to robust tumor formation and metastasis seen in mock-transfected PC3 cells. Similarly LNCaP cells expressing core3 O-glycans barely produced subcutaneous tumors in contrast to robust tumor formation by mock-transfected LNCaP cells. These findings indicate that addition of core3 O-glycans to beta1 and alpha2 integrin subunits in prostate cancer cells suppresses tumor formation and tumor metastasis.

Effects of Differential Glycosylation of Glycodelins on Lymphocyte Survival

Glycodelin is a human glycoprotein with four reported glycoforms, namely glycodelin-A (GdA), glycodelin-F (GdF), glycodelin-C (GdC), and glycodelin-S (GdS). These glycoforms have the same protein core and appear to differ in their N-glycosylation. The glycosylation of GdA is completely different from that of GdS. GdA inhibits proliferation and induces cell death of T cells. However, the glycosylation and immunomodulating activities of GdF and GdC are not known. This study aimed to use ultra-high sensitivity mass spectrometry to compare the glycomes of GdA, GdC, and GdF and to study the relationship between the immunological activity and glycosylation pattern among glycodelin glycoforms. Using MALDI-TOF strategies, the glycoforms were shown to contain an enormous diversity of bi-, tri-, and tetra-antennary complex-type glycans carrying Galbeta1-4GlcNAc (lacNAc) and/or GalNAcbeta1-4GlcNAc (lacdiNAc) antennae backbones with varying levels of fucose and sialic acid substitution. Interestingly, they all carried a family of Sda (NeuAcalpha2-3(GalNAcbeta1-4)Gal)-containing glycans, which were not identified in the earlier study because of less sensitive methodologies used. Among the three glycodelins, GdA is the most heavily sialylated. Virtually all the sialic acid on GdC is located on the Sda antennae. With the exception of the Sda epitope, the GdC N-glycome appears to be the asialylated counterpart of the GdA/GdF glycomes. Sialidase activity, which may be responsible for transforming GdA/GdF to GdC, was detected in cumulus cells. Both GdA and GdF inhibited the proliferation, induced cell death, and suppressed interleukin-2 secretion of Jurkat cells and peripheral blood mononuclear cells. In contrast, no immunosuppressive effect was observed for GdS and GdC.

Biochemical, Functional, and Pharmacological Characterization of AT-56, an Orally Active and Selective Inhibitor of Lipocalin-type Prostaglandin D Synthase

We report here that 4-dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)-butyl]-piperidine (AT-56) is an orally active and selective inhibitor of lipocalin-type prostaglandin (PG) D synthase (L-PGDS). AT-56 inhibited human and mouse L-PGDSs in a concentration (3–250 μm)-dependent manner but did not affect the activities of hematopoietic PGD synthase (H-PGDS), cyclooxygenase-1 and -2, and microsomal PGE synthase-1. AT-56 inhibited the L-PGDS activity in a competitive manner against the substrate PGH2 (Km = 14 μm) with a Ki value of 75 μm but did not inhibit the binding of 13-cis-retinoic acid, a nonsubstrate lipophilic ligand, to L-PGDS. NMR titration analysis revealed that AT-56 occupied the catalytic pocket, but not the retinoid-binding pocket, of L-PGDS. AT-56 inhibited the production of PGD2 by L-PGDS-expressing human TE-671 cells after stimulation with Ca2+ ionophore (5 μm A23187) with an IC50 value of about 3 μm without affecting their production of PGE2 and PGF2α but had no effect on the PGD2 production by H-PGDS-expressing human megakaryocytes. Orally administered AT-56 (<30 mg/kg body weight) decreased the PGD2 production to 40% in the brain of H-PGDS-deficient mice after a stab wound injury in a dose-dependent manner without affecting the production of PGE2 and PGF2α and also suppressed the accumulation of eosinophils and monocytes in the bronco-alveolar lavage fluid from the antigen-induced lung inflammation model of human L-PGDS-transgenic mice.

Activity, Splice Variants, Conserved Peptide Motifs, and Phylogeny of Two New α1,3-Fucosyltransferase Families (FUT10 and FUT11)

We report the cloning of three splice variants of the FUT10 gene, encoding for active alpha-l-fucosyltransferase-isoforms of 391, 419, and 479 amino acids, and two splice variants of the FUT11 gene, encoding for two related alpha-l-fucosyltransferases of 476 and 492 amino acids. The FUT10 and FUT11 appeared 830 million years ago, whereas the other alpha1,3-fucosyltransferases emerged 450 million years ago. FUT10-391 and FUT10-419 were expressed in human embryos, whereas FUT10-479 was cloned from adult brain and was not found in embryos. Recombinant FUT10-419 and FUT10-479 have a type II trans-membrane topology and are retained in the endoplasmic reticulum (ER) by a membrane retention signal at their NH(2) termini. The FUT10-479 has, in addition, a COOH-ER membrane retention signal. The FUT10-391 is a soluble protein without a trans-membrane domain or ER retention signal that transiently localizes to the Golgi and then is routed to the lysosome. After transfection in COS7 cells, the three FUT10s and at least one FUT11, link alpha-l-fucose onto conalbumin glycopeptides and biantennary N-glycan acceptors but not onto short lactosaminyl acceptor substrates as do classical monoexonic alpha1,3-fucosyltransferases. Modifications of the innermost core GlcNAc of the N-glycan, by substitution with ManNAc or with an opened GlcNAc ring or by the addition of an alpha1,6-fucose, suggest that the FUT10 transfer is performed on the innermost GlcNAc of the core chitobiose. We can exclude alpha1,3-fucosylation of the two peripheral GlcNAcs linked to the trimannosyl core of the acceptor, because the FUT10 fucosylated biantennary N-glycan product loses both terminal GlcNAc residues after digestion with human placenta alpha-N-acetylglucosaminidase.

Free light chains in plasma of patients with light chain amyloidosis and non‐amyloid light chain deposition disease. High proportion and heterogeneity of disulfide‐linked monoclonal free light chains as pathogenic features of amyloid disease

Immunoglobulin light chain amyloidosis (AL) and non-amyloid light chain deposition disease (NALCDD) are different forms of protein aggregation disorders accompanied by a monoclonal gammopathy. Monoclonal free light chains (FLCs) are precursors of the pathological light chain tissue deposits that are fibrillar in AL and granular in NALCDD. However, direct biochemical examination of plasma FLC precursors, which would allow comparison and better understanding of these two diseases, is still lacking. In this study, we examined FLCs in plasma of patients with AL and NALCDD by employing separation on Sep-PaK C18 cartridges, micro-preparative electrophoresis, Western blotting and mass spectrometry. Comparative analysis of AL versus NALCDD and control plasma samples showed new evidence of increased level and heterogeneity of circulating disulfide-bound FLC species in AL. In addition to full length monomers comprising the disulfide-linked FLCs, the monoclonal disulfide-bound FLC fragments were typically revealed in AL plasma. We hypothesized that enhanced disulfide binding of FLCs in AL interferes with their normal clearance and metabolism, which in turn might play a role in amyloid formation. The applied methods might be useful to diagnose or predict the pathological form of the disease and shed light on the mechanisms involved in light chain aggregation in tissues.

The NMR Structures of the Major Intermediates of the Two-domain Tick Carboxypeptidase Inhibitor Reveal Symmetry in Its Folding and Unfolding Pathways

There is a lack of experimental structural information about folding intermediates of multidomain proteins. Tick carboxypeptidase inhibitor (TCI) is a small, disulfide-rich protein consisting of two domains that fold and unfold autonomously through the formation of two major intermediates, IIIa and IIIb. Each intermediate contains three native disulfide bonds in one domain and six free cysteines in the other domain. Here we have determined the NMR structures of these two intermediates trapped and isolated at acidic pH in which they are stable and compared their structures with that of the native protein analyzed under the same conditions. Both IIIa and IIIb were found to contain a folded region that corresponds to the N- and C-terminal domains of TCI, respectively, with structures very similar to the corresponding regions of the native protein. The remainder of the polypeptide chains of the intermediates was shown to be unfolded in a random coil conformation. Solvent exchange measurements further indicated that the two protein domains are not completely independent, but affect each other in terms of dynamics and stability, in agreement with reported inhibitory activity data. The derived results provide structural evidence for symmetric TCI folding and unfolding mechanisms that converge in IIIa and IIIb and reveal the structural basis that accounts for the strong and simultaneous accumulation of both intermediates. Altogether, this work has important implications for a better understanding of the folding mechanisms of multidomain, disulfide-rich proteins.

Molecular Forms of BNP and ANP and gene expression of processing enzyme in atrium and ventricle in heart failure rat

Object: Recent studies suggest that there are several molecular forms of plasma BNP in patients with heart failure. However, precise mechanism remains unknown. Methods: The atrial and ventricular tissue levels of BNP1-45(alpha-BNP), BNP1-95(gamma-BNP), ANP1-28(alpha-ANP), and ANP1-126(gamma-ANP) were determined by radioimmunoassay following extraction with a Sep-Pak C18 cartridge and gel-filtration in control and Dahl salt-sensitive heart failure rat. We also measured mRNA levels of ANP, BNP, corin and furin, processing enzyme of ANP and BNP, in atrial and ventricular tissue by real-time PCR analysis.

Retinal Pigment Epithelium Lipofuscin Proteomics

Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein ( approximately 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E(2) adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.

A modified analytical method for the shipboard determination of nanomolar concentrations of orthophosphate in seawater

A method for the determination of nanomolar concentrations of orthophosphate in oligotrophic seawater developed by Liang et al. (2007) has been modified to make it fully feasible for shipboard application and for faster sample throughput with minimized sample volume. The technique is based on the flow injection method with solid phase extraction on a Sep-Pak C18 cartridge and colorimetric detector. The Schlieren effect was minimized by rinsing the cartridge sequentially with 5 mL water and 2 mL 95% ethanol solution. With three micro pumps in parallel, savings of up to 80% in amount of reagents and 25% volume of seawater samples could be achieved in comparison to the previous method. Variation of stopped flow time and sample loading time gave 3 different standard curves, which corresponded to 3 linear ranges within 3.4 and 515 nM. The modified method permits the analysis of samples over a wide range of concentrations, and has been successfully applied to shipboard determination of trace orthophosphate in more than 200 seawater samples during a one-month cruise in the South China Sea. For seawater at concentrations of 20.6, 82.5, 206.2 nM orthophosphate, the relative standard deviations (RSD) (n = 6), determined daily for 6 days on board ship were 4.45%, 4.73% and 6.75%, respectively. Five seawater samples collected in the Station SEATS (South East Asia Time Series Station at 18°N, 116°E) were analyzed using the present method both on board and in a land-based laboratory, as well as with the magnesium hydroxide-induced coprecipitation (MAGIC) method, and showed no significant difference according to the statistical t-test.