D6PK plasma membrane polarity requires a repeated CXX(X)P motif and PDK1-dependent phosphorylation.

D6 PROTEIN KINASE (D6PK) is a polarly localized plasma-membrane-associated kinase from Arabidopsis thaliana that activates polarly distributed PIN-FORMED auxin transporters. D6PK moves rapidly to and from the plasma membrane, independent of its PIN-FORMED targets. The middle D6PK domain, an insertion between kinase subdomains VII and VIII, is required and sufficient for association and polarity of the D6PK plasma membrane. How D6PK polarity is established and maintained remains to be shown. Here we show that cysteines from repeated middle domain CXX(X)P motifs are S-acylated and required for D6PK membrane association. While D6PK S-acylation is not detectably regulated during intracellular transport, phosphorylation of adjacent serine residues, in part in dependence on the upstream 3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE, promotes D6PK transport, controls D6PK residence time at the plasma membrane and prevents its lateral diffusion. We thus identify new mechanisms for the regulation of D6PK plasma membrane interaction and polarity.

Zinc complexes of chloroquine and hydroxychloroquine versus the mixtures of their components: Structures, solution equilibria/speciation and cellular zinc uptake.

The zinc complexes of chloroquine (CQ; [Zn(CQH+)Cl3]) and hydroxychloroquine (HO-CQ; [Zn(HO-CQH+)Cl3]) were synthesized and characterized by X-Ray structure analysis, FT-IR, NMR, UV-Vis spectroscopy, and cryo-spray mass spectrometry in solid state as well as in aqueous and organic solvent solutions, respectively. In acetonitrile, up to two Zn2+ ions bind to CQ and HO-CQ through the tertiary amine and aromatic nitrogen atoms (KN-aminCQ = (3.8 ±â€¯0.5) x 104 M-1 and KN-aromCQ = (9.0 ±â€¯0.7) x 103 M-1 for CQ, and KN-aminHO-CQ = (3.3 ±â€¯0.4) x 104 M-1 and KN-aromHO-CQ = (1.6 ±â€¯0.2) x 103 M-1 for HO-CQ). In MOPS buffer (pH 7.4) the coordination proceeds through the partially deprotonated aromatic nitrogen, with the corresponding equilibrium constants of KN-arom(aq)CQ = (3.9 ±â€¯1.9) x 103 M-1and KN-arom(aq)HO-CQ = (0.7 + 0.4) x 103 M-1 for CQ and HO-CQ, respectively. An apparent partition coefficient of 0.22 was found for [Zn(CQH+)Cl3]. Mouse embryonic fibroblast (MEF) cells were treated with pre-synthesized [Zn((HO-)CQH+)Cl3] complexes and corresponding ZnCl2/(HO-)CQ mixtures and zinc uptake was determined by application of the fluorescence probe and ICP-OES measurements. Administration of pre-synthesized complexes led to higher total zinc levels than those obtained upon administration of the related zinc/(hydroxy)chloroquine mixtures. The differences in the zinc uptake between these two types of formulations were discussed in terms of different speciation and character of the complexes. The obtained results suggest that intact zinc complexes may exhibit biological effects distinct from that of the related zinc/ligand mixtures.

Human serum albumin-bound selenium (Se-HSA) in serum and its correlation with other selenium species.

INTRODUCTION: Selenium (Se) is a trace element with different toxicological and nutritional properties according to its chemical forms. Among the wide range of selenium species, human serum albumin-bound selenium (Se-HSA) has still uncertain composition in terms of organic or inorganic selenium species. This study aimed at investigating the relation between Se-HSA levels with total selenium and the specific organic and inorganic selenium species. METHODS: We determined levels of total selenium and selenium species in serum of participants enrolled in two populations of the Emilia-Romagna region, in Northern Italy. Anion exchange chromatography coupled with inductively coupled plasma dynamic reaction cell mass spectrometry was used as quantification method. Correlations between Se-HSA and the other selenium compounds were analyzed using linear regression and restricted cubic spline regression models, adjusted for potential confounders. RESULTS: The first cohort comprised 50 participants (men/women: 26/24) with median (interquartile range, IQR) age 50 (55-62) years, while the second was composed of 104 participants (M/W: 50/54), median (IQR) age 48 (44-53) years. Median (IQR) levels of total selenium were 118.5 (109-136) µg/L and 116.5 (106-128) µg/L, respectively, while Se-HSA was 25.5 µg/L (16.2-51.5) and 1.1 (0.03-3.1) µg/L, respectively. In both populations, Se-HSA was positively associated with inorganic selenium species. Conversely, Se-HSA was inversely associated with organic selenium, especially with selenoprotein P-bound-Se (Se-SELENOP) and less strongly with selenomethionine-bound-Se (Se-Met), while the relation was null or even positive with other organic species. Evaluation of non-linear trends showed a substantially positive association with inorganic selenium, particularly selenite, until a concentration of 30 µg/L, above which a plateau was reached. The association with Se-SELENOP was inverse and strong until 100 µg/L, while it was almost null at higher levels. CONCLUSIONS: Our findings seem to indicate that Se-HSA incorporates more selenium when circulating levels of inorganic compounds are higher, thus supporting its mainly inorganic nature, particularly at high circulating levels of selenite.

Association of Urinary and Dietary Selenium and of Serum Selenium Species with Serum Alanine Aminotransferase in a Healthy Italian Population.

The trace element selenium is of considerable interest due to its toxic and nutritional properties, which markedly differ according to the dose and the chemical form. It has been shown that excess selenium intake increases the risk of type 2 diabetes and, possibly, other metabolic diseases like hyperlipidemia and non-alcoholic fatty liver disease (NAFLD). For the latter, however, epidemiologic evidence is still limited. We carried out a cross-sectional study recruiting 137 healthy blood donors living in Northern Italy and assessed their exposure to selenium through different methods and measuring serum selenium species. We performed linear and spline regression analyses to assess the relation of selenium and its forms with serum alanine aminotransferase (ALT) levels, a marker of NAFLD. Urinary selenium levels were positively and somewhat linearly correlated with ALT (beta regression coefficient (ß) 0.11). Conversely, the association of dietary selenium intake with ALT was positive up to 100 µg/day and null above that amount (ß 0.03). Total serum selenium was inversely associated with ALT up to 120 µg/L, and slightly positive above that amount. Concerning the different serum selenium species, ALT positively correlated with two organic forms, selenocysteine (ß 0.27) and glutathione peroxidase-bound selenium (ß 0.09), showed a U-shaped relation with the inorganic tetravalent form, selenite, and an inverse association with human serum albumin-bound selenium (ß -0.56). Our results suggest that overall exposure to selenium, and more specifically to some of its chemical forms, is positively associated with ALT, even at levels so far generally considered to be safe. Our findings add to the evidence suggesting that low-dose selenium overexposure is associated with NAFLD.

Accurate quantification of metal-glycinates-sulphate complexes and free metals in feed by capillary electrophoresis inductively coupled plasma mass spectrometry.

Traceability of metal-glycinate-sulphate complexes (Metal-GLY) in feed requires specific analysis to differentiate complexes from inorganic forms. A previously described method focused on the quantification of Metal-GLY at one single concentration but not on the quantification of free metal ion forms. The objective of this work was to extend the method to quantify both Metal-GLY and free metal ion forms of various metals at low inclusion levels. A 50/50 w/w mix of corn flour and soybean meal was used as feed. Copper-glycinate(Cu-GLY), Manganese-glycinate (Mn-GLY) and Zinc-glycinate (Zn-GLY) complexes (provided by Pancosma SA) were used for in-feed inclusions. The feed metal background concentrations and species repartitions were assessed. Cu-GLY was spiked on feed at levels matching 5, 15 and 45 mg/kg, corresponding to metal concentrations of 1.2, 3.6 and 10.8 mg/kg. Mn-GLY and Zn-GLY were spiked at 15, 45 and 100 mg/kg, corresponding to 3.3, 9.9, 22 mg/kg Mn and 3.9, 11.7, 26mg/kg Zn, respectively. The water soluble fraction of un-supplemented feed contained 0.06 mg/kg Cu, 0.05 mg/kg Mn and 0.12 mg/kg Zn, with 69.5% of Cu, 33.2% of Mn and 24.3% of Zn being present under free metal ions but 30.4% of Cu being present under Cu-GLY, 66.82% of Mn and 75.7% of Zn being present under Mn-GLY and Zn-GLY, respectively. The supplemented feeds at the 3 tested doses, from the lowest to the highest inclusion levels, contained in total respectively: 1.1, 3.05 and 9.06 mg/kg Cu; 2.99, 8.9 and 18.2 mg/kg Mn; 3.72, 10.9 and 23.4 mg/kg Zn. The M-GLY species recovered by analysis within the different supplemented feeds ranged from 76.26 to 89.32% for Cu-GLY, form 94.5 to 98.51% for Mn-GLY and from 76.05 to 98.96% for Zn-GLY. These results showed that CE-ICP-MS technique can be used to quantify low doses and to measure metal-species repartition between Metal-GLY and free metal ions, when included in feeds. For the first time, this study highlighted that the raw materials used contain Metal-GLY compounds. This raises the question of the occurrence of these compounds within the different raw materials used in feed production that could dramatically affect the way to supplement minerals in animal feed.

Elevated Levels of Selenium Species in Cerebrospinal Fluid of Amyotrophic Lateral Sclerosis Patients with Disease-Associated Gene Mutations.

BACKGROUND: Although an increasing role of genetic susceptibility has been recognized, the role of environmental risk factors in amyotrophic lateral sclerosis (ALS) etiology is largely uncertain; among neurotoxic chemicals, epidemiological and biological plausibility has been provided for pesticides, the heavy metal lead, the metalloid selenium, and other persistent organic pollutants. Selenium involvement in ALS has been suggested on the basis of epidemiological studies, in vitro investigations, and veterinary studies in which selenium induced a selective toxicity against motor neurons. OBJECTIVE: Hypothesizing a multistep pathogenic mechanism (genetic susceptibility and environmental exposure), we aimed to study selenium species in ALS patients carrying disease-associated gene mutations as compared to a series of hospital controls. METHODS: Using advanced analytical techniques, we determined selenium species in cerebrospinal fluid sampled at diagnosis in 9 ALS patients carrying different gene mutations (C9ORF72, SOD1, FUS, TARDBP, ATXN2, and TUBA4A) compared to 42 controls. RESULTS: In a patient with the tubulin-related TUBA4A mutation, we found highly elevated levels (in µg/L) of glutathione-peroxidase-bound selenium (32.8 vs. 1.0) as well as increased levels of selenoprotein-P-bound selenium (2.4 vs. 0.8), selenite (1.8 vs. 0.1), and selenate (0.9 vs. 0.1). In the remaining ALS patients, we detected elevated selenomethionine-bound selenium levels (0.38 vs. 0.06). CONCLUSIONS: Selenium compounds can impair tubulin synthesis and the cytoskeleton structure, as do tubulin-related gene mutations. The elevated selenium species levels in the TUBA4A patient may have a genetic etiology and/or represent a pathogenic pathway through which this mutation favors disease onset, though unmeasured confounding cannot be excluded. The elevated selenomethionine levels in the other patients are also of interest due to the toxicity of this nonphysiological selenium species. Our study is the first to assess selenium exposure in genetic ALS, suggesting an interaction between this environmental factor and genetics in triggering disease onset.

Redox speciation of iron, manganese, and copper in cerebrospinal fluid by strong cation exchange chromatography - sector field inductively coupled plasma mass spectrometry.

A new method of simultaneous redox speciation of iron (II/III), manganese (II/III), and copper (I/II) in cerebrospinal fluid (CSF) has been designed. For the separation of redox species strong cation exchange chromatography (SCX) with isocratic elution was employed. Species were detected using inductively coupled plasma sector field mass spectrometry (ICP-sf-MS), operating at medium resolution. The following parameters were optimized: analytical column, eluent composition and pH, CSF injection volume and dilution factor. Analytical column Dionex IonPac CS5A RFIC 4*250 mm was found to retain and separate species of interest the most effectively under the isocratic elution with a buffer, containing 50 mM ammonium citrate, 7.0 mM pyridine-2,6-dicarboxylic acid at pH = 4.2 and flow rate of 0.8 L min-1. Injection volume of 50 µL with CSF sample dilution of 1/3 (v/v) with the eluent was shown to result in minimal matrix suppression. For species identification, retention time matching with standards was used. The stability of metalloproteins (ferritin, transferrin, and ceruloplasmin) under elution conditions was evaluated. For the quantification of redox species, external calibration was employed. To avoid column contamination, a blank was run after measurement and all quantification values were blank subtracted. For recovery checks, species quantification data was verified against total content of an element, measured by dynamic reaction cell ICP-MS. Recoveries (sum of quantified species vs. total element determinations) were 82.5 ± 22% (Mn), 92 ± 11% (Fe), and 88.7 ± 12% (Cu). The method was tested using 38 real CSF samples. Limits of detection (3σ) for the CSF samples were 0.5 µg L-1, 0.6 µg L-1, and 0.8 µg L-1 for Fe, Mn, and Cu species, respectively. Retention time precision was 1-7.5% (as RSD), whereas peak area RSDs were in the range 5-11%, both depending on the species.

Determinants of serum manganese levels in an Italian population.

Manganese (Mn) is both essential and toxic for humans, mainly depending on the total levels and its species. Main sources of exposure include food and air pollution, particularly motorized traffic. We sought to determine the potential influence of these sources on serum total levels of Mn and Mn species. We selected a random sample of municipality residents from an Italian urban municipality, from whom we collected detailed personal information, dietary habits and a blood sample for serum Mn determination. We also assessed outdoor air Mn exposure, by modeling levels of particulate matter ≤10 µm (PM10) from motorized traffic at the residence of geocoded subjects. Serum Mn species generally showed higher levels in males and positive correlation with age, while no such differences were found according to smoking habits or use of dietary supplements. Among nutrients, only iron intake showed a relation with Mn [an inverse correlation with Mn­ferritin (Mn­Fer) and a direct one with inorganic­Mn (Inorg­Mn)]. Meat consumption directly correlated and fish and seafood inversely correlated with total Mn, Mn­transferrin (Mn­Tf) and Mn-citrate (Mn-Cit). Fruits and vegetables, including legumes and nuts, generally showed a positive correlation with all Mn species, especially Mn­Cit, and an inverse one with Inorg­Mn. Odds ratios (ORs) of having serum Mn levels above median value increased with increasing PM10 tertiles, with an OR for highest­to­lowest tertile of 7.40 (1.36­40.25) in multivariate analysis. Analyses for Mn species did not highlight a clear comparable pattern. In conclusion, our results seem to demonstrate that PM10 exposure positively influences total Mn serum levels, while single Mn species show conflicting results.

Toenail selenium as an indicator of environmental exposure: A cross-sectional study.

The relation between toxicity and essentiality of selenium (Se) is of growing interest in human health, as the effects may widely differ depending of its different chemical species and the exposure levels. Toenail Se has been proposed as a reliable biomarker of long-term Se exposure, but few studies investigated the correlation between its toenail content and environmental determinants (i.e., dietary food intake). We aimed to determine the relation of toenail Se levels with serum Se species as well as food items. We recruited a random sample of Modena (Northern Italy) municipal residents, from whom we collected detailed personal information, dietary habits, toenail specimen for Se determination and a blood sample for serum Se speciation analysis. Toenail Se mean value was 0.96 µg/g (range, 0.47­1.60), with slightly higher levels in females, in non-obese subjects and in Se supplements users, while it was lower in current smokers. Toenail Se positively correlated with organic Se forms, mainly selenoprotein P and selenocysteine, and inversely with the inorganic forms (selenite and selenate). Toenail Se was not associated with meat, cereals and dairy products consumption, positively correlated with fruit and slightly with vegetable intake, and negatively with fish and seafood consumption. Finally, no clear association emerged with estimated air Se exposure.

Lead, cadmium and mercury in cerebrospinal fluid and risk of amyotrophic lateral sclerosis: A case-control study.

Exposure to neurotoxic chemicals such as pesticides, selenium, and heavy metals have been suggested to play a role in the etiology of amyotrophic lateral sclerosis (ALS). We assessed exposure to lead, cadmium, and mercury in 38 ALS patients (16 men and 22 females) and 38 hospital-admitted controls by using their cerebrospinal fluid (CSF) content as biomarker. We determined CSF heavy metal levels with inductively coupled plasma sector field mass spectrometry, according to a methodology specifically developed for this biological matrix. ALS patients had higher median values for Pb (155 vs. 132ng/L) but lower levels for Cd (36 vs. 72ng/L) and Hg (196 vs. 217ng/L). In the highest tertile of exposure, ALS odds ratio was 1.39 (95% CI 0.48-4.25) for Pb, 0.29 (0.08-1.04) for Cd and 3.03 (0.52-17.55) for Hg; however, no dose-response relation emerged. Results were substantially confirmed after conducting various sensitivity analyses, and after stratification for age and sex. Though interpretation of these results is limited by the statistical imprecision of the estimates, and by the possibility that CSF heavy metal content may not reflect long-term antecedent exposure, they do not lend support to a role of the heavy metals cadmium, lead and mercury in ALS etiology.

Determinants of serum cadmium levels in a Northern Italy community: A cross-sectional study.

INTRODUCTION: Cadmium (Cd) is a heavy metal and a serious environmental hazard to humans. Some uncertainties still exist about major sources of Cd exposure in non-occupationally exposed subjects in addition to cigarette smoking, such as diet and outdoor air pollution. We sought to determine the influence of these sources on a biomarker of exposure, serum Cd concentration. METHODS: We recruited 51 randomly selected residents from an Italian urban community, from whom we obtained detailed information about dietary habits and smoking habits, and a blood sample for serum Cd determination. We also assessed outdoor air Cd exposure, by modeling outdoor air levels of particulate matter ≤10µm (PM10) from motorized traffic at geocoded subjects' residence. RESULTS: In crude analysis, regression beta coefficients for dietary Cd, smoking and PM10 on serum Cd levels were 0.03 (95% CI -0.83 to 0.88), 6.96 (95% CI -0.02 to 13.95) and 0.62 (95% CI -0.19 to 1.43), respectively. In the adjusted analysis, regression beta coefficients were -0.34 (95% CI -1-40 to 0.71), 5.81 (95% CI -1.43 to 13.04) and 0.47 (95% CI -0.35 to 1.29), respectively. CONCLUSION: Cigarette smoking was the most important factor influencing serum Cd in our non-occupationally exposed population, as expected, while dietary Cd was not associated with this biomarker. Outdoor air pollution, as assessed through exposure to particulate matter generated by motorized traffic, was an additional source of Cd exposure.

Selenium speciation in human serum and its implications for epidemiologic research: a cross-sectional study.

Observational studies addressing the relation between selenium and human health, particularly cancer risk, yielded inconsistent results, while most recent randomized trials showed a fairly consistent pattern suggesting null or adverse effects of the metalloid. One of the most plausible explanations for such inconsistencies is inadequate exposure assessment in observational studies, commonly carried out by measuring total Se content without taking into account the specific exposure to the individual chemical forms of the metalloid, whose toxic and nutritional properties may vary greatly. Data on the distribution of these species in human blood and their correlation with overall selenium levels are very limited. The concentrations of organic and inorganic selenium species were analyzed in serum of fifty subjects sampled from the general population of the municipality of Modena, northern Italy, aged from 35 to 70 years. Samples were collected during a 30-month period, and determinations of selenium species were carried out using high pressure liquid chromatography coupled with inductively coupled plasma dynamic reaction cell mass spectrometry. The majority of selenium was found to be present as organic species, but the inorganic forms showed higher levels than expected. These species showed limited correlations with age, sex and body mass index, while the organic forms increased in subjects consuming selenium-containing dietary supplements and decreased in smokers. The length of the sample storage period strongly influenced the distribution of selenium compounds, with a clear tendency towards higher inorganic and lower organic selenium levels over time. In multivariate analysis adjusting for potential confounders, total serum selenium correlated with human serum albumin-bound selenium and, in males, with two organic species of the metalloid (selenocysteine and glutathione peroxidase-bound selenium), while little association existed with the other organic forms and the inorganic ones. These findings highlight the potential for exposure misclassification of observational epidemiologic investigations based on overall selenium content in blood and possibly other tissues, and the critical role of the storage conditions for speciation analysis.

Study on selected trace elements and heavy metals in some popular medicinal plants from Sudan.

This study reports on the determination of 11 elements in 33 medicinal plants from Sudan and discusses a possible correlation between their curative effects and their trace elements content. Further, a possible accumulation of adverse heavy metals could be excluded. A total of 11 elements (cadmium, lead, mercury, tin, copper, iron, manganese, zinc, chromium, selenium and magnesium) were determined using inductively coupled plasma (ICP)-optical emission spectrometry (ICP-OES), ICP-sector field-mass spectrometry (ICP-sf-MS) and hydride generation (HG)-ICP-OES techniques. The results of the present study showed no heavy metal accumulation in any of the plants. Cd, Pb, Hg and Sn were found only in trace concentrations significantly below the global limits. This indicates the possibility of a safe use of these medicinal plants. Elevated chromium concentrations were found in those phytopharmaca which are employed for the treatment of diabetes mellitus in Sudanese traditional medicine. Cr was detected in the same range as in other plants reported to be applied for diabetes mellitus treatment. Aside from these medicinal plants, some others were identified which could be potential sources for providing reasonable amounts of Cr, Zn, Mn, Se and Mg for the treatment of diabetes mellitus, smooth muscle relaxation and/or against gastro-intestinal cramps.

Study on some trace element contents in serum and nail samples obtained from Sudanese subjects.

This study was performed to investigate trace elements and arsenic contents among Sudanese inhabitants living in the north, east, and west of Sudan. Inductively coupled plasma atomic emission spectrometry was used to determine the contents of Zn and Cu. Graphite furnace atomic absorption spectrometry was used to determine Se in serum samples. It was found that Se and Cu are in the normal range. Zinc showed discrepancies among all studied groups. Acute Zn deficiency was detected in the northern and eastern regions of Sudan. Inductively coupled plasma mass spectroscopy was operated in the dynamic reaction cell mode to determine the arsenic content in the nail samples of the northern inhabitants of Sudan. High values of arsenic were found in the northern people compared with the control group. This elevation could be linked to the misuse of insecticides and herbicides which might be associated with the high rate of cancer incidence in this region.

A method for low volume and low Se concentration samples and application to paired cerebrospinal fluid and serum samples.

The well-known beneficial health effects of Se have demanded the development of rapid and accurate methods for its analysis. A flow injection (FI) method with inductively coupled plasma mass spectrometry (ICP-MS) as a selenium-selective detector was optimized. Flow injection was carried out using a Knauer 1100 smartline inert series liquid chromatograph coupled with a Perkin Elmer DRC II ICP-mass spectrometer. For sample injection a Perkin Elmer electronic valve equipped with a 25microL sample loop was employed. Before measurement, standards or samples were administered with 1microg/L rhodium as internal standard for correction of changes in detector response according to changes in sample electrolyte concentration. The method characterization parameters are: LOD (3sigma criterion): 26ng/L, LOQ (10sigma criterion): 86ng/L, linearity: 0.05->10microg/L, r(2)=0.9999, serial or day-to-day precision at 2microg/L: 4.48% or 5.6%. Accuracy was determined by (a) recovery experiments (CSF spiked with 2microg/L Se); (b) comparison of FI-ICP-MS measurement with graphite furnace atomic absorption (GFAAS) measurements of 1:10 diluted serum samples; (c) Se determination in urine and serum control materials. Recovery (a) was 101.4%, measurement comparison with GFAAS (b) showed 98.8% (5 serum samples, 1:10 diluted in the range of 0.5-1.3microg/L, compared to GFAAS determination, which was set to 100%), and accuracy was 96.8% or 105.6% for the serum or urine control material. Analysis time per sample was short and typically below 2min for the complete measurement, including sample introduction, sample-line purge and quadruplicate Se determination. This method was used to determine Se in cerebrospinal fluid (CSF) and plasma (here parallel to GFAAS) in 35 paired serum and CSF samples. Se determination gave values in the range of 42-130microg/L for serum and 1.63-6.66microg/L for CSF. The median for Se in 35 individual CSF samples was 3.28microg/L, the mean (+/-SD) was 3.67 (1.35)microg/L, whilst for individual serum samples the median was 81microg/L and the mean (+/-SD) was 85 (26)microg/L. When relating the paired Se concentrations of CSF samples to respective serum samples it turned out that Se-CSF (behind blood brain barrier (BBB)) is independent on Se-serum concentration (before BBB).