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PubMed1999

Trans-speciation maintenance in the MHC region of a polymorphism which includes a polymorphic dinucleotide locus, and the de novo arisal of a polymorphic tetranucleotide microsatellite.

Alleles and the surrounding regions of DQCAR, a dinucleotide repeat tightly linked to HLA-DQB1, were sequenced in a range of primate species including man. Three polymorphic regions can usefully be defined in the description of these sequences: the dinucleotide GT repeat itself, the anonymous region 5' of this repeat, and a variable CTGT repeat in the 3' region. The 5' sequence displayed six alleles in the individuals studied. One of these alleles was invariably associated with substitutions in the GT repeat and absence of the CTGT repeat, the others with pure, polymorphic GT repeats and variation in the numbers of CTGT repeats. Haplotypes can be classified by the allele in the 5' region. Those carrying allele 1 were only found in man, those with allele 2 in man, chimpanzee and gorilla. The third haplotype (indicated by the presence of allele 3) was found in chimpanzee, gorilla and orang-utan, the fourth in chimpanzee and gibbon, the fifth in baboon, guenon and mangabey and the sixth in guenon and macaque. The alleles in the 5' region, but from different species, are thus often more similar than alleles from the same species, a phenomenon already shown for some HLA genes. This suggests that major histocompatibility sequences and surrounding sequences shared a correlated evolutionary history. The new polymorphic tetranucleotide microsatellite (CTGT, 3rd region) has possibly arisen de novo from the pre-existing dinucleotide GT. This study provides information not only on the molecular evolution of this particular microsatellite but also of the trans-speciation maintenance of polymorphism of its surrounding sequences.

Alleles↗
PubMed1988

MHC polymorphism pre-dating speciation.

Two features distinguish the polymorphism of the major histocompatibility complex (MHC) loci from that of other loci: its high diversity and the large genetic distance between MHC alleles. More than 100 alleles exist in natural populations in the mouse at each of the functional class I and class II alleles, all alleles occurring at frequencies that cannot be explained by recurrent mutations. Some of the alleles differ by approximately 70 nucleotides in the coding region alone and some of the products of the allelic genes differ by more than 50 amino acids. It has generally been assumed that these differences accumulated after species inception. Here, we present evidence for an alternative explanation of the origin of MHC polymorphism: a large part of the MHC polymorphism pre-dates speciation and is passed on from species to species. We describe allelic differences that must have arisen before the separation of mice and rats from a common ancestor more than 10 million years ago.

Alleles↗
PubMed1989

Adaptation, speciation and hybrid zones.

Many species are divided into a mosaic of genetically distinct populations, separated by narrow zones of hybridization. Studies of hybrid zones allow us to quantify the genetic differences responsible for speciation, to measure the diffusion of genes between diverging taxa, and to understand the spread of alternative adaptations.

Adaptation, Physiological↗
PubMed1992

Genetic divergence, speciation and morphological stasis in a lineage of African cichlid fishes.

Since their discovery at the turn of the century, the species assemblages of cichlid fishes in the East African Lakes Victoria, Malawi and Tanganyika have fascinated evolutionary biologists. Many models have attempted to account for the 'explosive' evolution of several hundred species within these lakes. Here we report a case of surprisingly large genetic divergence among populations of the endemic Tropheus lineage of Lake Tanganyika. This lineage of six species contains twice as much genetic variation as the entire morphologically highly diverse cichlid assemblage of Lake Malawi and six times more variation than the Lake Victoria species flock. Although it is highly variable in coloration, this group of species and its closest relatives have not undergone appreciable morphological change. The observed geographic pattern of genetic variation suggests that major lake level fluctuations affected the distribution and speciation of this lineage of cichlid fishes.

Africa↗
PubMed1997

Speciation of arsenic animal feed additives by microbore high-performance liquid chromatography with inductively coupled plasma mass spectrometry.

Phenylarsonic compounds have been used as poultry and swine feed additives for the purpose of growth promotion and disease prevention. Owing to the lack of suitable analytical methods, however, knowledge of their metabolism, environmental fate and impact remains incomplete. In order to compensate for this, analytical procedures were developed that allow the speciation of arsenic animal feed additives by using microbore high-performance liquid chromatography (microHPLC) coupled on-line with ICP-MS. More specifically, reversed-phase (RP) chromatographic methods were optimised to achieve the separation of various phenylarsonic acids from each other and from the more toxic inorganic arsenic compounds. This mode of chromatography, however, exhibits limitations, especially in the presence of naturally occurring organoarsenic compounds. The application of RP ion-pairing chromatography eliminates such shortcomings by minimising the co-elution of arsenic species. In general, the microHPLC-ICP-MS methods developed in this study provide high selectivity, extremely good sensitivity, low limits of detection (low-ppb or sub-pg amounts of As), require small sample volumes (< 1 microliter), minimise waste and operate most efficiently under low mobile-phase flow rates (15-40 microliters min-1), which are compatible for use with other types of mass spectrometers, e.g., electrospray. Reference materials containing naturally occurring arsenic compounds were spiked with phenylarsonic compounds and then analysed by using the procedures developed in this study.

Animal Feed↗
PubMed1998

Chemical speciation of arsenic in serum of uraemic patients.

Chemical speciation of arsenic was carried out in serum of a total of 51 uraemic patients: 19 non-dialysis (ND), 18 haemodialysis (HD) and 14 continuous ambulatory peritoneal dialysis (CAPD) patients. The low molecular mass As species were separated by ion-exchange liquid chromatography and measured on-line by hydride generation atomic absorption spectrometry (HGAAS). The high molecular mass As species were separated by fast protein liquid chromatography, either size-exclusion, ion-exchange or affinity chromatography, and the fractions were digested and measured off-line with HGAAS. The mean total As concentrations in the serum of the three groups of the uraemic patients were significantly higher than the reference value (6.47 +/- 4.28, 5.12 +/- 5.58 and 4.67 +/- 5.41 micrograms l-1 for HD, ND and CAPD patients, respectively, versus the reference value of 0.96 +/- 1.52 micrograms l-1. The major As species in serum of the patients were dimethylarsinic acid (DMA) and arsenobetaine. The HD patients showed a significantly higher mean DMA level than ND and CAPD patients. No selective removal of different As species in serum of HD patients was observed after 4 h of haemodialysis. The inorganic As species in serum were bound to proteins, mainly transferrin (about 5-6% of total As in serum). This binding may play an important role in arsenic detoxification.

Adult↗
PubMed1998

Speciation of basal aluminium in human serum by fast protein liquid chromatography with inductively coupled plasma mass spectrometric detection.

The coupling of fast protein liquid chromatography (FPLC) with inductively couples plasma mass spectrometry (ICP-MS) was evaluated as a technique for studying aluminium bound to proteins present in human serum. Separation of human serum proteins was achieved on a MonQ (HR5/5) anion-exchange column using an ammonium acetate gradient (0-0.25 mol I-1) at the physiological pH of 7.4 (0.05 mol l-1 TRIS-HC1 buffer). Aluminium contamination was avoided with an on-line Al-chelating scavenger column. Proteins were detected spectrophotometrically at 295 nm and the Al detection was carried out on-line using both quadrupole ICP-MS and double-focusing ICP-MS systems. At metal basal levels in serum the latter detector proved to be adequate for this detection. Results obtained with the procedure developed confirmed clearly that transferrin is the only significant A1-binding proteins in unspiked uraemic serum. In addition, a high-resolution ICP-MS instrument was applied successfully as an A1-specific detector allowing for the first time A1 speciation studies in unspiked normal serum. The technique can also be used for studying the protein binding of elements other than A1.

Aluminum↗
PubMed1998

Speciation of selenium in the mammalian organism.

As almost all of the selenium present in the mammalian organism is protein-bound, speciation is mostly concerned with the determination of the different selenium-containing proteins. Information on their distribution and their concentrations in the different tissues of the rat was obtained by means of tracer procedures which, after application of 75Se-selenite with a very high specific activity to selenium-depleted animals and electrophoretic separation of the labelled proteins, allow the determination of these compounds in the pmol to fmos range. A method was developed for the determination of selenocysteine and selenomethionine in the selenium-containing proteins. The identification of specific selenoproteins was achieved by analysis of their selenoamino acid residues and by studies on their characteristics and possible biological functions. This is being followed by the development of methods for the quantitative analysis of the selenoproteins in questions in the tissues of animals and man. In this paper the strategies and procedures used in the identification, characterization and determination of the selenium species present in the mammalian organism will be discussed.

Animals↗
PubMed1998

Speciation determination of arsenic in urine by high-performance liquid chromatography-hydride generation atomic absorption spectrometry with on-line ultraviolet photooxidation.

A coupled system for arsenic speciation determination based on high-performance liquid chromatography (HPLC), on-line UV photooxidation and continuous-flow hydride generation atomic absorption spectrometry (HGAAS) was built from commercially available modules with minor modifications to the electronic interface, the software and the gas-liquid separator. The best results were obtained with strong anion-exchange columns, Hamilton PRP X-100 and Supelcosil SAX 1, and gradient elution with phosphate buffers containing KH2PO4-K2HPO4. The on-line UV photooxidation with alkaline peroxodisulfate, 4% m/v K2S2O8-1 mol l-1 NaOH, in a PTFE knotted reactor for 93 s ensures the transformation of inorganic AsIII, monomethylarsonate, dimethylarsinate, arsenobetaine, arsenocholine, trimethylarsine oxide and tetramethylarsonium ion to arsenate. About 32-36 HPLC-UV-HGAAS runs could be performed within 8 h, with limits of detection between 2 and 6 micrograms l-1 As, depending on the species. The method was applied to the analysis of spot urine samples and certified urine reference materials (CRMs). Upon storage at 4 degrees C, reconstituted CRMs are stable for at least 2 weeks with respect to both their total arsenic content and the individual species distribution.

Arsenic↗
PubMed1998

Speciation measurements by HPLC-HGAAS of dimethylarsinic acid and arsenobetaine in three candidate lyophilized urine reference materials.

Speciation measurements of dimethylarsinic acid (DMA) and arsenobetaine (AsB) in three candidate lyophilized urine reference materials are described. The measurements were based on cation-exchange liquid chromatography coupled to hydride generation atomic absorption spectrometry with on-line digestion of the organic. As species by alkaline persulfate solution aided by ultraviolet radiation. Arsenic concentrations as DMA were significantly different in the three samples. The mean values for the three samples were 4.1 +/- 0.3, 55.3 +/- 1.2 and 134.1 +/- 1.5 micrograms l-1, respectively. No significant differences in AsB concentrations were observed among the three samples. The mean As concentrations as AsB in the three samples were 17.4 +/- 0.4, 17.7 +/- 0.2 and 17.5 +/- 0.3 micrograms l-1, respectively. By off-line digestion of the urine samples, total As concentrations in the three materials were also obtained. The mean values were 23.4 +/- 0.3, 76.6 +/- 1.6 and 151.3 +/- 1.8 micrograms l-1, respectively. These results correlated well with the results obtained by neutron activation analysis in our laboratory (r = 0.999; p < 0.0001).

Arsenicals↗
PubMed1999

Speciation of metabolites of selenate in rats by HPLC-ICP-MS.

The metabolic pathway for and metabolites of selenium (Se) administered intravenously to rats in the form of selenate at a dose of 0.3 mg Se kg-1 body weight were studied by speciating Se in the bloodstream, liver and urine by HPLC-inductively coupled argon plasma mass spectrometry. Selenate was not taken up by red blood cells (RBCs) and disappeared from the bloodstream much faster than selenite, without any change in its chemical form before it disappeared from the plasma. Selenium excreted into the urine after the administration of selenate showed different patterns from those of selenite in both amounts and chemical forms. With the selenate group, the concentration of Se in urine was highest at 0-6 h and the chemical species of Se was selenate at 0-6 h; thereafter a monomethylselenol-related Se compound (MMSe*) and trimethylselenonium ions (TMSe) appeared, selenate not being excreted after 6 h. On the other hand, in the selenite group, the concentration of Se peaked at 6-12 h, and the chemical species of Se were MMSe* and TMSe. Selenate was reduced in vitro on incubation in either a liver homogenate or supernatant fraction, although much more slowly than in the whole body. Selenate was not reduced by glutathione or dithiothreitol. The results suggest that in contrast to selenite, which is taken up by and reduced in RBCs, and then transferred to the liver, approximately 20% of the selenate administered to rats was excreted into the urine without any change in its chemical form with the present dose, and the major portion of selenate was taken up by the liver, reduced and then utilized for the synthesis of selenoproteins or excreted into the urine after being methylated.

Animals↗
PubMed1999

Ensuring quality in long term environmental monitoring for chemical speciation.

The awareness regarding quality assurance (QA) and quality control (QC) for environmental monitoring has considerably increased in the past few years, especially with respect to the determination of chemical species, since errors occurring at various levels may considerably affect the interpretation of results (e.g. studies of toxic impact, geochemical pathways, etc.). QA for environmental analysis covers a broad range of activities from sample collection to laboratory work and the approach for ensuring quality data should be considered in a global context. However, whereas great emphasis has been placed on QA within the laboratory, there have been few systematic attempts to evaluate risks of discrepancies related, for example, to field manipulations. The situation is even worse when monitoring is applied to relatively new fields such as chemical speciation. This paper outlines some of the main aspects of quality control of environmental analysis, including the validation of methods, sampling and sample handling, storage, etc., with special emphasis on the monitoring of chemical forms of elements (e.g. species of tin, mercury, lead and selenium).

Chemistry Techniques, Analytical↗
PubMed2000

Selenium speciation in enriched and natural samples by HPLC-ICP-MS and HPLC-ESI-MS with perfluorinated carboxylic acid ion-pairing agents.

Selenium-enriched plants, such as hyperaccumulative phytoremediation plants (Astragalus praleongus, 517 micrograms g-1 Se, and Brassica juncea, 138 micrograms g-1 Se in dry sample), yeast (1200, 1922 and 2100, micrograms g-1 Se in dry sample), ramp (Allium tricoccum, 48, 77, 230, 252, 405 and 524 micrograms g-1 Se in dry sample), onion (Allium cepa, 96 and 140 micrograms g-1 Se in dry sample) and garlic (Allium sativum, 68, 112, 135, 296, 1355 micrograms g-1 Se in dry sample) were analyzed by HPLC-ICP-MS for their selenium content and speciation after hot water and enzymatic extractions. Reference samples with natural selenium levels, such as onion and garlic controls, cooking garlic powder, baking yeast powder and a commercial garlic supplement were also analyzed. Selected samples were also examined by HPLC-electrospray ionization (ESI)-MS. HPLC was mostly carried out with 0.1% heptafluorobutanoic acid (HFBA) as ion-pairing agent in 1 + 99 v/v methanol-water solution, but 0.1% trifluoroacetic acid (TFA) in 1 + 99 v/v methanol-water solution was also utilized to permit chromatography for compounds that did not elute with HFBA. More than 75% of the total eluting selenium compounds, based upon element specific detection, were identified from retention time data and standard spiking experiments, and between 60 and 85% of compounds were identified by MS, with up to 25% of the total eluting molecular selenium species being unidentified as yet. Limits of quantification (LOQ, defined as the concentration giving an S/N of 10) for HPLC-ICP-MS were in the range 2-50 ng mL-1 Se in the injected extracts for the selenium-enriched samples and 2-10 ng mL-1 Se for the natural selenium level samples. LOQ values for HPLC-ESI-MS were ca. 100 times higher than those measured by HPLC-ICP-MS.

Chromatography, High Pressure Liquid↗
PubMed1989

Application of high-performance liquid chromatography--inductively coupled plasma mass spectrometry to the investigation of cadmium speciation in pig kidney following cooking and in vitro gastro-intestinal digestion.

The speciation of cadmium in retail pig kidney has been examined by size-exclusion chromatography (SEC) coupled directly to inductively coupled plasma mass spectrometry (ICP-MS). Approximately 35% of the cadmium from uncooked kidney was soluble after aqueous extraction at pH 8 and SEC - ICP-MS revealed three discrete peaks whose retention times corresponded to estimated relative molecular masses of 1.2 x 10(6), 7.0 x 10(4) and 6 x 10(3)-9 x 10(3). In the cooked kidney, 35% of the Cd was soluble and was all associated with a peak of a relative molecular mass (Mr) of 6 x 10(3)-9 x 10(3). After simulated gastric digestion of cooked pig kidney at pH 2.5, 60% of the cadmium was solubilised and associated with a species of Mr less than 1 x 10(3). When the digest was also subjected to simulated intestinal digestion at pH 6.8, a single peak, which corresponded to 20% of the original cadmium, was eluted. This peak co-eluted with the single peak extracted at pH 8.0 from the cooked kidney. It was also of similar estimated Mr to the single broad peak observed after simulated gastro-intestinal digestion of equine renal metallothionein (Mr = 1.1 x 10(4]. The results suggest that the majority of soluble cadmium in retail pig kidney is associated with a metallothionein-like protein that survives both cooking and simulated in vitro gastro-intestinal digestion.

Animals↗
PubMed1992

Utilization of two different chemical forms of selenium during lactation using stable isotope tracers: an example of speciation in nutrition.

The bioavailability and metabolism of different chemical species of mineral nutrients in the diet are receiving much attention from research nutritionists. In order to make scientifically based recommendations for mineral intakes, the chemical form of the mineral, with its specific absorption, utilization and retention, needs to be considered. Selenium is an example of an essential nutrient that is consumed in several different chemical forms, hence information is needed on the bioavailability and metabolism of each form before recommendations for dietary intakes can be made. A valuable tool for research on bioavailability and metabolism in humans is stable isotope tracers. When there are more than two stable isotopes available, as with selenium, stable isotope methodology allows the comparison of the utilization of different chemical forms of the nutrient simultaneously in in vivo studies. As an example of speciation questions addressed by nutritionists, a study is described that simultaneously evaluated utilization (absorption, retention and appearance in milk and blood) of two different chemical forms of selenium (selenite and selenomethionine) in lactating, non-lactating and never pregnant women using stable isotope tracers. All three groups of women had similar selenium status at the start of the study. Significantly more selenium from selenomethionine than from selenite was absorbed and appeared in the plasma in all groups. Milk contained more selenium from apparently absorbed selenomethionine than from selenite. All groups retained significantly more selenium from selenomethionine than from selenite; lactating women retained more selenium from selenite than did the other two groups, suggesting that milk losses may be partially compensated by enhanced retention of dietary selenium as selenite.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Size, morphology and composition of particulates in aquatic ecosystems: solving speciation problems by correlative electron microscopy.

Particulates can impact directly on aquatic ecosystems by determining the availability and mode of dispersion of both contaminants and nutrients. An understanding of the mechanisms of such particle-associated phenomena is being augmented by particle analysis technology. In this context, microscopic and spectroscopic techniques, devised for problem solving, are being applied to frequently encountered sub-micrometre particulates which are 'unstable' with respect to methods of sample preparation and storage used routinely for particulates prior to analysis. These unstable aquatic particulates include 'species' sensitive to dehydration and to artificial aggregation induced by surfaces within a fractionation apparatus. These species, as defined broadly, include polysaccharide gels, hydrated humic substances, iron oxyhydroxides, viruses, the smallest micro-organisms and decomposing parts of cells. To develop predictive models of their roles as dispersing agents for contaminants, and to speciate such associations, it is necessary to characterize them in a state as close to the natural as possible. This critical review presents the state-of-the-art in the realistic characterization of hydrated sub-micrometre particulates by correlative electron microscopy (EM) used in conjunction with spectroscopy and minimally perturbing preparatory techniques. Correlative EM is a strategy for using several different kinds of microscopes and accessory techniques in a multi-method context to analyse a given specimen for different kinds of information, including relationships in three dimensions within colloid systems. Sizing, morphology and gross composition are determined on a 'per particle' basis by transmission EM used in conjunction with energy-dispersive spectroscopy, electron diffraction and molecule-specific stains.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Simultaneous speciation of butyltin and phenyltin compounds in the waters of south-west Spain.

An analytical method for the simultaneous speciation of butylin and phenyltin compounds has been applied to a study of the water quality in six locations (rivers and sea) in South West Spain. It was not intended to provide a comprehensive survey of the area but to establish what the maximum levels might be for a subsequent and more detailed study. Phenyltin species were not found in any sample, however tributyltin was present (100-400 ng l-1) in the Cadiz Bay samples, which suggests that the biological water quality may sometimes be depressed. Wherever tributyltin was present, dibutyltin and monobutyltin occurred, possibly, as a result of a degradation process.

Chromatography, Gas↗