Indices of iron homeostasis in asymptomatic subjects with HFE mutations and moderate ferritin elevation during iron removal treatment.

We analysed iron biomarkers and their relationships in 30 subjects with HFE mutations and moderate hyperferritinaemia undergoing iron removal at our blood donation centre. Body mass index (BMI) and liver enzymes were assessed. Serum iron (SI), ferritin, transferrin saturation (TSAT), hepcidin and non-transferrin bound iron (NTBI) were measured serially. Seventeen subjects had p.C282Y/p.C282Y, nine p.C282Y/p.H63D, four p.H63D/p.H63D. Median age (p = 0.582), BMI (p = 0.500) and ferritin (p = 0.089) were comparable. At baseline, 12/17 p.C282Y/p.C282Y and 2/9 p.C282Y/p.H63D had measurable NTBI (p = 0.003). The p.C282Y/p.C282Y had higher TSAT (p < 0.001), lower hepcidin (p = 0.031) and hepcidin/ferritin ratio (p = 0.073). After treatment, iron indices were similar among groups, except TSAT (higher in p.C282Y/p.C282Y; p = 0.06). Strong relationships were observed between ferritin and TSAT (R = 0.71), NTBI and TSAT (R = 0.61), NTBI and SI (R = 0.54) in p.C282Y/p.C282Y. Hepcidin correlated weakly with ferritin in p.C282Y/p.C282Y (R = 0.37) but strongly in p.C282Y/p.H63D (R = 0.66) and p.H63D/p.H63D (R = 0.72), while relationships with TSAT were weak (R = 0.27), moderate (R = 0.55) and strong (R = 0.61), respectively. Low penetrance p.C282Y/p.C282Y phenotype displays hepcidin dysregulation and biochemical risk for iron toxicity.

ß-Amyloid in Cerebrospinal Fluid: How to Keep It Floating (Not Sticking) by Standardization of Preanalytic Processes and Collection Tubes.

BACKGROUND: Phosphorylated tau (pTau), total tau (tTau), and ß-amyloid (Aß) are established cerebrospinal fluid (CSF) biomarkers used to help diagnose Alzheimer disease. Preanalytic workups of CSF samples lack harmonization, making interlaboratory comparison of these biomarkers challenging. The Aß adsorbs to sample tubes, yielding underestimated concentrations, and may result in false Alzheimer disease diagnosis. Our primary aim was to compare Aß recovery across multiple polypropylene tubes and to test the stability of tTau, pTau, and Aß in the best performing tube. METHODS: Eight polypropylene tubes were tested using 3 CSF pools with Aß concentrations <500, 500-1000, and >1000 ng/L. All samples were analyzed in duplicate. Tubes were cut open to assess their different infrared adsorption spectra. Freshly drawn CSF from 14 patients was distributed into 4 Sarstedt 5-mL (no. 63.504.027; Sar5CSF) tubes, left at room temperature for up to 7 days, and analyzed for pTau, tTau, and Aß by ELISA. RESULTS: Two Sarstedt 5-mL tubes and a Sarstedt 10-mL (Sar10CSF) tube showed significantly higher Aß recovery at all 3 concentrations compared with the 5 other tubes. The infrared adsorption spectra of Sar10CSF and Sar5CSF tubes were practically identical, unlike the other tubes. No significant loss of pTau, tTau, and Aß was observed in CSF left at room temperature for up to 7 days (P > 0.05). CONCLUSIONS: Recovery of Aß from Sar5CSF tubes is equivalent to Aß recovery from Sar10CSF tubes. Levels of pTau, tTau, and Aß were stable for at least 7 days at room temperature but not at 37 °C.

Cytokine and Gene Expression Profiling in Patients with HFE-Associated Hereditary Hemochromatosis according to Genetic Profile.

BACKGROUND: Hemochromatosis gene (HFE)-associated hereditary hemochromatosis (HH) is characterized by downregulation of hepcidin synthesis, leading to increased intestinal iron absorption. OBJECTIVES: The objectives were to characterize and elucidate a possible association between gene expression profile, hepcidin levels, disease severity, and markers of inflammation in HFE-associated HH patients. METHODS: Thirty-nine HFE-associated HH patients were recruited and assigned to 2 groups according to genetic profile: C282Y homozygotes in 1 group and patients with H63D, as homozygote or in combination with C282Y, in the other group. Eleven healthy first-time blood donors were recruited as controls. Gene expression was characterized from peripheral blood cells, and inflammatory cytokines and hepcidin-25 isoform were quantified in serum. Biochemical disease characteristics were recorded. RESULTS: Elevated levels of interleukin 8 were observed in a significant higher proportion of patients than controls. In addition, compared to controls, gene expression of ζ-globin was significantly increased among C282Y homozygote patients, while gene expression of matrix metalloproteinase 8, and other neutrophil-secreted proteins, was significantly upregulated in patients with H63D. CONCLUSION: Different disease signatures may characterize HH patients according to their HFE genetic profile. Studies on larger populations, including analyses at protein level, are necessary to confirm these findings.

Kidney stone compositions and frequencies in a Norwegian population.

Objectives: The aim of this study was to investigate frequencies of kidney stone constituents in a Norwegian population and examine trends over time by comparing with an earlier study of this population. Materials and methods: Upper urinary tract calculi consecutively collected from patients who underwent stone surgery at Akershus University Hospital from July 2014 to December 2017, in total 1252 calculi, were analysed by infrared spectroscopy. The results were compared with a study of 500 calculi collected from June 1975 to September 1980 at the same hospital. Results: The male:female ratio was 1.83:1. Single-component stones constituted 39%, 35% were binary, and 25% ternary. Main stone component frequencies were oxalate 71.3%, calcium oxalate monohydrate 53.7% with higher occurrence in males, calcium oxalate dihydrate 17.6%, carbonate apatite 10.8% and struvite 5.7%, both with higher occurrence in females, uric acid 8.9% with a non-significant male predominance, brushite 1.6% and cystine about 1%. Over four decades the frequency of UA stones increased by 4.6-times, whereas struvite and pure carbonate apatite stones decreased and no change was observed for brushite stones. Conclusion: Frequencies of kidney stone types in this Norwegian population are mainly in accordance with other studies, except a large increase in UA stones over four decades, partly caused by a particularly low frequency of UA stones in the old study, a decreased carbonate apatite frequency over four decades, and an unaltered brushite frequency. Also, in contrast to other studies, a relatively small and non-significant male UA stone predominance was found.

Paralithocins, Antimicrobial Peptides with Unusual Disulfide Connectivity from the Red King Crab, Paralithodes camtschaticus.

As part of an ongoing exploration of marine invertebrates as a source of new antimicrobial peptides, hemocyte extracts from the red king crab, Paralithodes camtschaticus, were studied. Three cationic cysteine (Cys)-rich peptides, named paralithocins 1-3, were isolated by bioassay-guided purification, and their amino acid sequences determined by Edman degradation and expressed sequences tag analysis. Disulfide bond mapping was performed by high-resolution tandem mass spectrometry. The peptides (38-51 amino acids in length) share a unique Cys motif composed of eight Cys, forming four disulfide bridges with a bond connectivity of (Cys relative position) Cys1-Cys8, Cys2-Cys6, Cys3-Cys5, and Cys4-Cys7, a disulfide arrangement that has not been previously reported among antimicrobial peptides. Thus, paralithocins 1-3 may be assigned to a previously unknown family of antimicrobial peptides within the group of Cys-rich antimicrobial peptides. Although none of the isolated peptides displayed antimicrobial activity against the target strains Escherichia coli, Pseudomonas aeruginosa, or Staphylococcus aureus, they inhibited the growth of several marine bacterial strains with minimal inhibitory concentrations in the 12.5-100 µM range. These findings corroborate the hypothesis that marine organisms are a valuable source for discovering bioactive peptides with new structural motifs.

Secretoneurin is a novel prognostic cardiovascular biomarker associated with cardiomyocyte calcium handling.

BACKGROUND: Secretoneurin (SN) levels are increased in patients with heart failure (HF), but whether SN provides prognostic information and influences cardiomyocyte function is unknown. OBJECTIVES: This study sought to evaluate the merit of SN as a cardiovascular biomarker and assess effects of SN on cardiomyocyte Ca(2+) handling. METHODS: We assessed the association between circulating SN levels and mortality in 2 patient cohorts and the functional properties of SN in experimental models. RESULTS: In 143 patients hospitalized for acute HF, SN levels were closely associated with mortality (n = 66) during follow-up (median 776 days; hazard ratio [lnSN]: 4.63; 95% confidence interval: 1.93 to 11.11; p = 0.001 in multivariate analysis). SN reclassified patients to their correct risk strata on top of other predictors of mortality. In 155 patients with ventricular arrhythmia-induced cardiac arrest, SN levels were also associated with short-term mortality (n = 51; hazard ratio [lnSN]: 3.33; 95% confidence interval: 1.83 to 6.05; p < 0.001 in multivariate analysis). Perfusing hearts with SN yielded markedly increased myocardial levels and SN internalized into cardiomyocytes by endocytosis. Intracellularly, SN reduced Ca(2+)/calmodulin (CaM)-dependent protein kinase II δ (CaMKIIδ) activity via direct SN-CaM and SN-CaMKII binding and attenuated CaMKIIδ-dependent phosphorylation of the ryanodine receptor. SN also reduced sarcoplasmic reticulum Ca(2+) leak, augmented sarcoplasmic reticulum Ca(2+) content, increased the magnitude and kinetics of cardiomyocyte Ca(2+) transients and contractions, and attenuated Ca(2+) sparks and waves in HF cardiomyocytes. CONCLUSIONS: SN provided incremental prognostic information to established risk indices in acute HF and ventricular arrhythmia-induced cardiac arrest.

Identification of peptide products from enzymatic degradation of amyloid beta.

Deposition of insoluble amyloid plaques is one of the known hallmarks of Alzheimer's disease. Amyloid beta 1-42 is the main component in these plaques, and the soluble oligomers of this peptide are believed to contribute to synaptic degradation and dementia. Enzymatic hydrolysis of amyloid beta is important to keep its tissue concentration low to avoid oligomerization. We have employed four enzymes involved in in vivo degradation of amyloid beta, to identify amyloid beta 1-42 hydrolysis products in vitro. Liquid chromatography coupled to (high resolution) mass spectrometry was used to identify the proteolysis products. Novel cleavage sites were discovered for all four enzymes. For each enzyme, the peptide was incubated for several different periods from 0.5 to 210 min, and the proteolysis products from each period were characterized. Thus, both the initial cleavage sites and the full degradation profiles were revealed. Knowledge about the fate of amyloid beta is important to better understand the mechanism underlying Alzheimer's disease, and the reported proteolysis products can be used as targets in future investigations on amyloid beta clearance.

The structure of the fire fighting foam surfactant Forafac®1157 and its biological and photolytic transformation products.

For several decades, perfluorooctane sulfonate (PFOS) has widely been used as a fluorinated surfactant in aqueous film forming foams used as hydrocarbon fuel fire extinguishers. Due to concerns regarding its environmental persistence and toxicological effects, PFOS has recently been replaced by novel fluorinated surfactants such as Forafac®1157, developed by the DuPont company. The major component of Forafac®1157 is a 6:2 fluorotelomer sulfonamide alkylbetaine (6:2 FTAB), and a link between the trade name and the exact chemical structure is presented here to the scientific community for the first time. In the present work, the structure of the 6:2 FTAB was elucidated by (1)H, (13)C and (19)F nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. Moreover, its major metabolites from blue mussel (Mytilus edulis) and turbot (Scophthalmus maximus) and its photolytic transformation products were identified. Contrary to what has earlier been observed for PFOS, the 6:2 FTAB was extensively metabolized by blue mussel and turbot exposed to Forafac®1157. The major metabolite was a deacetylated betaine species, from which mono- and di-demethylated metabolites also were formed. Another abundant metabolite was the 6:2 fluorotelomer sulfonamide. In another experiment, Forafac®1157 was subjected to UV-light induced photolysis. The experimental conditions aimed to simulate Arctic conditions and the deacetylated species was again the primary transformation product of 6:2 FTAB. A 6:2 fluorotelomer sulfonamide was also formed along with a non-identified transformation product. The environmental presence of most of the metabolites and transformation products was qualitatively demonstrated by analysis of soil samples taken in close proximity to an airport fire training facility.

Metabolism of small antimicrobial ß(2,2)-amino acid derivatives by murine liver microsomes.

We have investigated the in vitro metabolism of three small antimicrobial ß(2,2)-amino acid derivatives (M (w) < 500) that are highly potent against methicillin resistant Staphylococcus aureus, and are among the first compounds designed from small cationic antimicrobial peptides with potential for oral administration. The ß(2,2)-amino acid derivatives are virtually completely resistant against degradation by proteases, and to further explore their drug potential, we have investigated the hepatic Phase I metabolism of this class of antimicrobial compounds. The ß(2,2)-amino acid derivatives were incubated with murine liver microsomes and the metabolites analyzed semi-quantitatively by HPLC-MS and qualitatively by ultra performance liquid chromatography coupled to a tandem mass spectrometer which enabled identification of the metabolites by careful interpretation of the collision activated dissociation spectra. The study shows that sterically hindered ß(2,2)-amino acid derivatives that otherwise are stable against proteolytic degradation underwent Phase I metabolism and were oxidized to a number of different metabolites depending on the structure of the ß(2,2)-amino acid side-chains.

Plasma concentrations of parabens in postmenopausal women and self-reported use of personal care products: the NOWAC postgenome study.

Parabens are used extensively in personal care products; however, their estrogenic properties have raised concern over risks to human health. High levels of total parabens, mainly as conjugates, have been reported in human plasma/serum, with limited data on native parabens. Our objective was to assess and link plasma concentrations of native common parabens to self-reported use of personal care products in women from the general population. The information was obtained from an extensive questionnaire on diet and lifestyle previously answered by the women in the NOWAC study. Plasma samples from 332 individuals were extracted and cleaned up by automated solid phase extraction and analyzed by ultra high performance liquid chromatography time-of-flight mass spectrometry. Native methyl paraben dominated and was detected in 63% of the samples, with a median level of 9.4 ng/ml. Ethyl paraben (median < 3 ng/ml) and propyl paraben (median < 2 ng/ml) were detected in 22 and 29%, respectively. Butyl and benzyl parabens were not detected. For the first time, elevated levels of native parabens are reported in women from the general population. The concentrations were significantly associated with the use of skin lotions, indicating that frequent (daily or more) use maintain elevated concentrations despite the parabens short half-lives. These findings clearly emphasize the need to study potential health effects in the general population.

Centrocins: isolation and characterization of novel dimeric antimicrobial peptides from the green sea urchin, Strongylocentrotus droebachiensis.

As immune effector molecules, antimicrobial peptides (AMPs) play an important role in the invertebrate immune system. Here, we present two novel AMPs, named centrocins 1 (4.5kDa) and 2 (4.4kDa), purified from coelomocyte extracts of the green sea urchin, Strongylocentrotus droebachiensis. The native peptides are cationic and show potent activities against Gram-positive and Gram-negative bacteria. The centrocins have an intramolecular heterodimeric structure, containing a heavy chain (30 amino acids) and a light chain (12 amino acids). The cDNA encoding the peptides and genomic sequences were cloned and sequenced. One putative isoform (centrocin 1b) was identified and one intron was found in the genes coding for the centrocins. The full length protein sequence of centrocin 1 consists of 119 amino acids, whereas centrocin 2 consists of 118 amino acids which both include a preprosequence of 51 or 50 amino acids for centrocins 1 and 2, respectively, and an interchain of 24 amino acids between the heavy and light chain. The difference of molecular mass between the native centrocins and the deduced sequences from cDNA indicates that the native centrocins contain a post-translational brominated tryptophan. In addition, two amino acids at the C-terminal, Gly-Arg, were removed from the light chains during the post-translational processing. The separate peptide chains of centrocin 1 were synthesized and the heavy chain alone was shown to be sufficient for antimicrobial activity. The genome of the closely related species, the purple sea urchin (S. purpuratus), was shown to contain two putative proteins with high similarity to the centrocins.

Perfluorinated compounds in maternal serum and cord blood from selected areas of South Africa: results of a pilot study.

There is limited information about both environmental and human perfluorinated compounds (PFCs) concentrations in the southern hemisphere, and for the first time, concentrations of these compounds are reported in maternal serum and cord blood of South African women. The majority of the participants were of African Black ethnicity, with a similar socioeconomic status. In maternal serum perfluorooctane sulfonate (PFOS) was found to be the most abundant PFC (1.6 ng mL(-1)), followed by perfluorooctanoate (PFOA: 1.3 ng mL(-1)) and perfluorohexane sulfonate (PFHxS: 0.5 ng mL(-1)); however, in cord blood PFOA was the most abundant compound (1.3 ng mL(-1)) followed by PFOS (0.7 ng mL(-1)) and PFHxS (0.3 ng mL(-1)). Linear PFOS constituted 58% of the sum of PFOS, comparable with a reported percentage from Australia. Differences in PFC concentrations between communities were found, with the highest concentrations in urban and semi-urban areas. The median maternal PFOS concentration was lower than has been reported in other studies, whereas the PFOA concentration was the same. This clearly indicates that the exposure pathway is different from the western world. Significant differences in housing quality were observed and the urban and sub-urban community had the highest living and housing standards. Possible exposure pathways could be different from those elucidated in the western world with the exception of the urban community in our study that showed higher living standards in general and easier access to modern consumer products.

Separation of DNA-containing organelles from Toxoplasma gondii by CZE.

Toxoplasma gondii and other members of the family Apicomplexa have two organelles, in addition to the nucleus, that contain DNA. Herein is reported the separation of the DNA-carrying organelles from T. gondii tachyzoites, i.e. the mitochondrion and the apicoplast, by CZE. The cells were stained with SYTO9, a dye that exhibit fluorescence when interacting with double stranded nucleic acids (e.g. DNA) and disrupted by nitrogen cavitation. Following careful removal of the heavier cellular material, the remaining lysate was injected on a CE instrument and the DNA-containing organelles were detected by LIF. The mitochondrion had longer migration time than the apicoplast, and the migration times were comparable in the replicates. This method should potentially also work for other members of the Apicomplexa including Plasmodium falciparum.

Total structure characterization of unsaturated acidic phospholipids provided by vicinal di-hydroxylation of fatty acid double bonds and negative electrospray ionization mass spectrometry.

In the present work, the unsaturated fatty acid substituents of some phosphatidic acid, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol species were converted to their 1,2-di-hydroxy derivatives by OsO(4). The subsequent electrospray ionization tandem low-energy mass spectrometry analysis of the deprotonated species allowed positional determination of the double bonds by the production of specific product-ions. The product-ions are formed by charge-remote and charge-proximate homolytic cleavages as well as charge-directed heterolytic cleavages and rearrangements. The commercial availability of pure species of the phospholipids in question was limited, and a number of species were therefore synthesized. The developed method was used to fully characterize the two isobaric phosphatidylglycerol species 16:0/16:1Delta(9) and 16:0/16:1Delta(10) extracted from the bacteria Methylococcus capsulatus. The presence of these fatty acids was supported by a gas-chromatography mass spectrometry investigation of the dimethyloxazoline derivatives of the species of the lipid extract. The present work demonstrates that a total structure characterization of acidic unsaturated phospholipids in isolate or in mixtures is accomplished by vicinal di-hydroxylation of olefinic sites and subsequent electrospray ionization mass spectrometry of the derivatized phospholipids.

Vicinal hydroxylation of unsaturated fatty acids for structural characterization of intact neutral phospholipids by negative electrospray ionization tandem quadrupole mass spectrometry.

We report a novel method allowing the complete structural characterization of intact species of the phospholipid classes phosphatidylcholine and phosphatidylethanolamine by utilizing negative electrospray ionization quadrupole tandem mass spectrometry (MS/MS). Information on the molecular weight of the intact phospholipid species, the class to which it belongs, the molecular mass of the fatty acid substituents and their regioisomerism, is easily revealed by MS/MS. Throughout our investigations the R2COO- ions were more abundant than the R1COO- ions, and this observation is used for regioisomeric assignment of the two fatty acids. However, for phospholipid species containing an unsaturated fatty acid, information on the position of the double bond is not achieved in this way. By converting the olefinic sites to their 1,2-dihydroxylated derivatives, information on the position of the hydroxyl groups (and hence of the double bond) is obtained by performing a second MS/MS experiment. Thus, a complete structural characterization of intact phosphatidylcholine and phosphatidylethanolamine species is obtained by performing these two MS/MS experiments. In order to ensure structural distinction of isobaric species, a number of phosphatidylethanolamine and phosphatidylcholine species were synthesized from lyso-phosphatidylcholine and analyzed by the present method. The applicability of the method to real samples is also demonstrated by the complete structural elucidation of the two phosphatidylcholine species 16:0/18:1Delta9 and 16:0/18:1Delta11 from egg yolk.

Negative electrospray ionization low-energy tandem mass spectrometry of hydroxylated fatty acids: a mechanistic study.

Recently, we reported that by converting olefinic fatty acids to their saturated vicinally 1,2-di-hydroxylated derivatives, abundant ions indicative for hydroxyl group locations are produced by negative electrospray ionization low-energy tandem mass spectrometry, allowing the assignment of the olefinic site in the native fatty acid. In this report the mechanisms whereby the characteristic ions are produced are investigated. The mono-hydroxylated fatty acid, 12-hydroxyoctadecanoic acid, served as a model for the more complex 12,13-dihydroxyoctadecanoic acid, and fragmentation mechanisms accounting for the most abundant product ions generated from their deprotonated molecules are proposed. In general, three different mechanisms are proposed to operate in the formation of the observed product ions: (i) step-wise charge-remote homolytic cleavages, (ii) step-wise charge-proximate homolytic cleavages, and (iii) concerted charge-directed rearrangement reactions involving bond formation(s) and heterolytic cleavages. Support for the proposed mechanisms was achieved by investigating the deuterium- and oxygen-18-labeled isotopomers of both compounds.

Structure elucidation of unsaturated fatty acids after vicinal hydroxylation of the double bonds by negative electrospray ionisation low-energy tandem mass spectrometry.

A method for determining the positions of double bonds in unsaturated fatty acids by use of negative electrospray ionisation low-energy tandem mass spectrometry is described. First, a vicinal hydroxylation of the double bonds of mono- and poly-unsaturated fatty acids was performed. Low-energy collision activation dissociation of the deprotonated molecules produced structurally informative ions formed by a-cleavages relative to the hydroxyl groups. Abundant fragment ions that confirmed the positions of all hydroxyl groups, and thus the positions of the double bonds in the native fatty acids, were observed in the spectra of derivatised mono-, di-, and tri-unsaturated fatty acids. Two types of ions were observed, called [alpha'(n)](-) and [alpha(n)](-). The letter n indicates the positions of the hydroxyl groups. The structurally diagnostic ions [alpha'(n)](-) were produced by cleavages distal to the hydroxyl-groups with the charge retained on the carboxylate. [alpha'(n)](-) ions originating from all hydroxyl-groups were observed in the spectra of modified mono-, di-, and tri-unsaturated fatty acids. Initial proton transfer of a hydroxyl proton to the carboxylate with subsequent cleavages proximal to the hydroxyl groups, relative to the carboxylate, resulted in the two structurally diagnostic [alpha(n)](-) ions. In hydroxylated fatty acids having two or more double bonds in their native structure, [alpha(n)](-) ions originating only from the two final hydroxyl-groups were observed. The formation of all ions of [alpha'(n)](-) and [alpha(n)](-) type can be rationalised by a six-membered transition state. Hydroxylated deprotonated tetra-, penta-, and hexa-unsaturated fatty acids also produced [alpha'(n)](-) ions indicating the positions of most of the hydroxyl-groups, whereas the [alpha(n)](-) ions were observed as described above. The method described offers a simple approach to the determination of the positions of double bonds in unsaturated fatty acids, and is an alternative to utilising di-lithiated fatty acids. The method should also be applicable when fatty acids are constituents of more complex molecules such as phospholipids.