Determination of the neuropeptides arginine vasotocin and isotocin in brains of three-spined sticklebacks (Gasterosteus aculeatus) by off-line solid phase extraction-liquid chromatography-electrospray tandem mass spectrometry.

A method based on solid phase extraction (SPE) followed by liquid chromatography-electrospray ionisation tandem mass spectrometry for the determination of the nonapeptides arginine vasotocin (AVT) and isotocin (IT) in brains of three-spined sticklebacks (Gasterosteus aculeatus) is described. Separation and detection were optimized using synthetic standards. Limits of detection (LOD) for standard solutions were 160 pg mL(-1) for AVT and 250 pg mL(-1) for IT. The SPE procedure hardly affected the LODs for standard solutions. Mainly because of ion suppression, LODs for AVT and IT in brains were approximately 5 and 25 pg mg(-1), respectively. The concentrations determined in the brain of several fishes ranged from 10 to 500 pg mg(-1) for AVT and from 400 to 4000 pg mg(-1) for IT.

Properties and Units in the Clinical Laboratory Sciences.

This document describes the introduction of the concept of property in the field of clinical and environmental human toxicology for the presentation of results of clinical laboratory investigations. It follows the IFCC-IUPAC systematic terminological rules and attempts to create a common base for communication between the clinical chemist, the medical practitioner, the human toxicologist, and the environmental toxicologist. The term designating a substance being a toxicant may be an international nonproprietary name (INN), a generic name, a registered trade name, a fantasy name, or other. This causes difficulties in the transmission of requests and reports on properties involving such substances in biological fluids and environmental media to and from laboratories, to the end user, and in the collating of this information from different sources. The document comprises a list of properties of human and environmental systems involving toxicants for use in transmitting medical laboratory data. The document recommends terms based on the format developed by the IFCC and IUPAC to facilitate interaction between disciplines and unambiguous interpretation of data, e.g. for purposes of risk interpretation. Systematic terms are presented together with a code (identified by the letters NPU) for each. The complete C-NPU Database may be found at: http://dior.imt.liu.se/C-NPU.

Selenium speciation from food source to metabolites: a critical review.

Especially in the last decade, a vast number of papers on Se and its role in health issues have been published. This review gives a brief, critical overview of the main analytical findings reported in these papers. Of particular interest is the Se content in different food sources worldwide and the extent to which their consumption is reflected in the Se content of human tissues and body fluids. Several food sources, both natural (Brazil nuts, garlic, Brassica juncea) and Se-enriched (yeast-based supplements), are discussed as to origin, characteristics, Se metabolism and impact of their consumption on the human body. The continuous development of new and improvement of existing analytical techniques has provided different powerful tools to unravel the Se species and their function. An up-to-date literature study on Se speciation analysis is given, illustrating how analytical chemistry in its different facets aids in the identification of Se compounds and provides insight into the complete metabolic pathway of Se throughout the human body. This review includes a detailed image of the current state-of-the-art of Se speciation analysis in these food sources and in human tissues and body fluids.

Liquid chromatography-mass spectrometry (LC-MS): a powerful combination for selenium speciation in garlic (Allium sativum).

Liquid chromatography (LC) hyphenated with both elemental and molecular mass spectrometry has been used for Se speciation in Se-enriched garlic. Different species were separated by ion-pair liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS) after hot-water extraction. They were identified by on-line reversed-phase liquid chromatography-electrospray ionization tandem mass spectrometry (RPLC-ESI-MS-MS). Se-methionine and Se-methylselenocysteine were determined by monitoring their product ions. Another compound, gamma-glutamyl-Se-methylselenocysteine, shown to be the most abundant form of Se in the garlic, was determined without any additional sample pre-treatment after extraction and without the need for a synthesized standard. Product ions for this dipeptide were detected by LC-ESI-MS-MS for three isotopes of Se-78 Se, 80Se: and 82Se. The method was extended to the species extracted during in-vitro gastrointestinal digestion. Because both Se-methylselenocysteine and gamma-glutamyl-Se-methylselenocysteine have anticarcinogenic properties, their extractability and stability during human digestion are very important. Garlic was also treated with saliva, to enable detection and analysis of species extracted during mastication. Detailed information on the extractability of selenium species by both simulated gastric and intestinal fluid are given, and variation of the distribution of Se among the different species with time is discussed. Although the main species in garlic is the dipeptide gamma-glutamyl-Se-methylselenocysteine, Se-methylselenocysteine is the main compound present in the extracts after treatment with gastrointestinal fluids. Two more, so far unknown compounds were observed in the chromatogram. The extracted species and their transformations were analysed by combining LC-ICP-MS and LC-ESI-MS-MS. In both the simulated gastric and intestinal digests, Se-methionine, Se-methylselenocysteine, and gamma-glutamyl-Se-methylselenocysteine could be determined by LC-ESI-MS-MS by measuring their typical product ions.

Identification of the major selenium compound, Se-Methionine, in three yeast (Saccharomyces cerevisiae) dietary supplements by on-line narrowbore liquid chromatography-electrospray tandem mass spectrometry.

On-line monitoring of six Se-compounds was accomplished by using an XTerra MS C18 column coupled to electrospray tandem mass spectrometry (ES-MS-MS). In view of the nature of the compounds, the positively charged ion pairing agent tetraethylammoniumchloride (TEACl) was added to the mobile phase. The HPLC-ES-MS-MS method was optimized with six commercially available Se-compounds. Substitution of the analytical column by the narrowbore type significantly enhanced the sensitivity of the method. We were able to detect the m/z of these six molecules on-line. Furthermore, all product ions could be monitored. The method was applied to three different yeast-based supplements. They were submitted to proteolytic digestion and screened for their Se-content by HPLC-HG-AFS (hydride generation-atomic fluorescence spectrometry). By application of on-line narrowbore HPLC-electrospray tandem mass spectrometry, the main compound present in these three supplements, Se-Methionine, could be measured on its m/z and its product ions. The method can be further extended for on-line measurement of different Se-species in complex matrices

In vivo distribution and fractionation of indium in rats after subcutaneous and oral administration of [(114m)In]InAs.

Two in vivo experiments were carried out in this study. In the first experiment five rats were given two subcutaneous injections of [(114m)In]InAs. Major sites of accumulation were spleen, liver and kidney. The intracellular distribution of indium was examined by differential centrifugation. The cytoplasmic fraction contained most of the indium activity followed by the mitochondrial fraction. Both outcomes are in close agreement with the results obtained in previous studies. Chromatographic separations on a preparative size exclusion column were carried out. It was shown that indium was mostly bound to high molecular mass compounds in serum and in the cytoplasmic fraction of spleen, liver and kidney. In a second experiment five rats were given four oral doses of [(114m)In]InAs over a short period. Prior to this experiment the in vitro solubility of cold InAs in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) was determined using graphite furnace atomic absorption spectroscopy. In the case of the SGF only 1.3% of an InAs suspension dissolved after 48 hours incubation at 37 degrees C. InAs was not soluble in SIF. Uptake of InAs after oral administration was minimal (<1%). Due to incomplete removal of traces of [(114m)In]InAs from the gastrointestinal tract, it was impossible to calculate accurately the in vivo distribution over the different organs. As the uptake and consequently the activity in the organs were very low, no further chromatographic separations could be carried out. Considering this very low uptake, it can be concluded that InAs will not accumulate in the body after oral exposure.

Hyphenated techniques for speciation of Se in in vitro gastrointestinal digests of Saccharomyces cerevisiae.

A method was developed allowing the separation, detection and identification of Se species extracted from yeast supplements during simulated digestion processes. The in vitro gastric and intestinal digests were studied for their Se compounds by successive high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) and high-performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ES-MS-MS) analyses. The conditions for the separation were chosen as to be compatible with both ICP-MS and ES-MS-MS detection. HPLC-ICP-MS was used to screen the extracts for their Se content. By means of HPLC-ES-MS-MS, the compounds extracted were identified on-line according to their retention time, m/ z of the molecular ion and the presence of typical product ions. From these results, it was clear that the main compound extracted by both gastric and intestinal fluid was Se-methionine, which was also the main Se compound extracted by proteolytic digestion from the yeast supplements. Two other minor compounds could be identified as Se-cystine and Se(O)-methionine, a degradation product of Se-methionine.

Detection of metals in proteins by means of polyacrylamide gel electrophoresis and laser ablation-inductively coupled plasma-mass spectrometry: application to selenium.

The capabilities of laser ablation-inductively coupled plasma-mass spectrometry for the detection of trace elements in a gel after gel electrophoresis were systematically studied. Figures of merit, such as limit of detection, linearity, and repeatability, were evaluated for various elements (Li, V, Cr, Mn, Ni, Cu, Zn, As, Se, Mo, Pd, Ag, Cd, Pt, Tl, Pb). Two ablation strategies were followed: single hole drilling, relevant for ablation of spots after two-dimensional (2-D) separations, and ablation with translation, i.e., on a line, relevant for one-dimensional (1-D) separations. This technique was applied to the detection of selenoproteins in red blood cells extracts after a 1-D separation (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and the detection of selenium-containing proteins in yeast after 2-D electrophoresis (2-DE). The detection procedure was further improved by using the dynamic reaction cell technology, which allowed the removal of the Ar_2(+) interference and hence the use of the most abundant Se isotope, (80)Se. Reaction gases were compared (methane, carbon monoxide, ammonia, oxygen and the combination of argon (collision gas) and hydrogen (reaction gas)). In each instance, the reaction cell parameters were optimized in order to obtain the lowest detection limit for Se (as (80)Se(+), (82)Se(+) or (77)Se(+); and as (80)Se(16)O(+), (82)Se(16)O(+) or (77)Se(16)O(+) with O(2) as the reaction gas). Carbon monoxide was found to offer the best performance. The detection limit with the use of DRC and He as transport gas was 0.07 microg Se g(-1) gel with single hole drilling and 0.15 microg Se g(-1) gel for ablation with translation.

Safety evaluation of organoarsenical species in edible Porphyra from the China Sea.

A study was carried out to determine arsenic species in Porphyra seaweed originating from the China Sea. Information about arsenic species in Porphyra was provided by HPLC-ICP-MS and ES-MS-MS. The total arsenic concentrations of Porphyra samples from five different producing areas ranged from 2.1 to 21.6 mg/kg. The analysis report also showed that arsenosugars were the only arsenic species that could be detected in all of the extracts of samples. Arsenosugar PO(4) was the major compound in most samples (0.3-13.9 mg/kg of dry weight), followed by arsenosugar OH (0.7-6.2 mg/kg of dry weight). A further experiment was done to investigate the stability of arsenosugars in the process of being heated. It was observed that the arsenosugars were stable during a short-term heating at 100 degrees C. Their stability in human ingestion was also studied. A substantial increase of dimethylarsinic acid (DMA) was detected in urine samples collected from six volunteers after the consumption of this seaweed. The results obtained indicated that arsenosugars had been metabolized to DMA, which is more toxic than arsenosugars. From this point of view, consumers should consider the possible adverse effects of edible Porphyra on human health and choose those Porphyra having lower arsenic concentrations.

A survey of arsenic species in Chinese seafood.

In the present report, thirty different types of Chinese edible seafood, including brown algae, red algae, fish, crab, shrimp, mussels, oysters, and clams, which are very popular foodstuffs in the Chinese kitchen, were examined for their total content of As as well as its different species. Total arsenic concentration in algae samples was 1.7-38.7 microg/g (dry weight), and 0.086-7.54 microg/g in fish and shellfish (wet weight), respectively. The arsenic species in seafood extracts were determined by using anion and cation exchange high performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICPMS). Arsenosugars were detected in all of the extracted algae samples (1.5-33.8 microg/g dry weight) and fish samples (0.018-0.78 microg/g wet weight). Arsenobetaine was detected in all of the extracted fish and shellfish samples (0.025-6.604 microg/g wet weight). In contrast, inorganic arsenic in fish and shellfish samples occurred at levels below 2% of the total arsenic. No inorganic arsenic was detected in the algae samples. This study provides information about the distribution pattern of arsenic species in seafood products. Since the major share of arsenic components in seafood is organic arsenic with a low toxicity, we can conclude that arsenic in seafood does not pose any risk to human health.

Urinary excretion of the uraemic toxin p-cresol in the rat: contribution of glucuronidation to its metabolization.

BACKGROUND: Increasing evidence indicates that lipophilic and/or protein-bound substances such as p-cresol are responsible for adverse physiological alterations in uraemic patients. To better understand the evolution of p-cresol disposition in renal failure and dialysis patients, it is necessary to determine its kinetic characteristics and biotransformation pathways. METHODS: We studied the biotransformation of p-cresol after intravenous injection of the compound in eight rats with normal renal function. Urine was collected in four 1 h intervals. To evaluate the presence of p-cresol metabolites, beta-glucuronidase was added to urine samples and the isolated unidentified chromatographic peak observed in previous experiments was submitted to tandem mass spectrometry (MS/MS) analysis. RESULTS: Administration of p-cresol produced a p-cresol peak and an unknown peak, suggesting biotransformation of the compound. Addition of beta-glucuronidase to urine samples and incubation at 37 degrees C resulted in a marked decrease in the unidentified peak height (P<0.001) together with an increase in p-cresol peak height (P<0.001), suggesting that the unidentified peak was composed, at least in part, of p-cresylglucuronide. Mass spectrometry (MS) and MS/MS analysis of the isolated unidentified peak confirmed the presence of p-cresylglucuronide. Linear regression between the peak height of p-cresylglucuronide before enzyme treatment and the increase in p-cresol peak height after enzyme treatment in samples incubated with beta-glucuronidase allowed us to calculate the amount of p-cresylglucuronide as its p-cresol equivalents. This revealed that 64% of the injected p-cresol was excreted as glucuronide. There was no change in peak heights when sulphatase was added to the urine. When p-cresol and p-cresylglucuronide levels were combined, approximately 85% of all administered p-cresol was recovered in the urine. In addition, the combined urinary excretion of p-cresol and p-cresylglucuronide was more than four times greater than excretion of p-cresol by itself (P<0.01). CONCLUSIONS: In rats with normal renal function, intravenous administration of p-cresol results in immediate and extensive metabolization of the compound into p-cresylglucuronide. The elimination of p-cresol from the body depends largely on the urinary excretion of this metabolite.

Vanadium speciation in serum by means of blue native gel electrophoresis.

Slab-gel electrophoresis has been applied to the speciation of vanadium in serum. The electrophoresis separation is an adaptation of the blue native polyacrylamide gel electrophoresis separation necessary to ensure the stability of the vanadium-protein complex; Coomassie blue was used to shift the charges of the proteins and to stabilize the vanadium complex. The detection of the vanadium species was made possible by the use of the (48)V radiotracer and the phosphor-screen technology. The method was first developed using transferrin, incubated with (48)V, as a model. After it was proved that the vanadium-transferrin complex was stable during separation, the method was validated by separating serum incubated with (48)V. The efficiency of the separation was assessed according to two parameters: resolution and conservation of the species. First, the resolution of the separation was as expected from a native separation. Second, the release of free vanadium from the transferrin complex, which was the main vanadium species expected, was negligible, which proves that the species remain intact during separation. In accordance with the literature, it was found that vanadium binds to transferrin in incubated serum at these low concentrations.

Fractionation of vanadium complexes in serum, packed cells and tissues of Wistar rats by means of gel filtration and anion-exchange chromatography.

Male Wistar rats were intraperitoneally injected with [(48)V]vanadium tracer to (1) investigate the distribution of vanadium over different tissues and (2) study the distribution of vanadium over the proteins and peptides in serum, packed cells and homogenates of tissues by means of liquid chromatography experiments (size exclusion, ion exchange). Target organs were primarily kidney, bone, spleen and liver. In serum we found that vanadium was mainly bound to transferrin; however, a small amount was also bound to albumin. Besides these two complexes, a significant part of vanadium occurred as readily exchangeable ("free") vanadium. In packed cells, vanadium is mainly bound to hemoglobin and to two abundant low molecular mass complexes. The chromatograms of tissues (kidney, liver, testes, spleen and lung) show similar high molecular mass complexes (vanadium co-elutes with ferritin, transferrin and hemoglobin). Between the low molecular mass complexes there are similar peaks for spleen, testes and kidneys on the one hand, and liver and lung on the other hand, albeit the differences are small. In the case of lung, there is an additional low molecular mass peak.

Behaviour of vanadate and vanadium-transferrin complex on different anion-exchange columns. Application to in vivo 48V-labelled rat serum.

The behaviour of free [48V]vanadate and [48V]vanadium-transferrin complex was investigated on five different anion-exchange columns (Mono Q 5/5 HR, Hitrap Q HP, Sepharose Q FF, Sepharose DEAE FF and Hitrap Q XL). The recovery of both V-compounds was quantitative. The peak shape and retention time of vanadate varied according to the type of column. The vanadium-transferrin complex also showed different elution patterns depending on the type of column. Especially in case of the Sepharose Q FF, Mono Q 5/5 HR and Hitrap Q XL columns the vanadium-transferrin binding was degraded during elution on the column. The results clearly prove that care should be taken as to the choice of column for speciation purposes of vanadium compounds in order to prevent various artefacts showing up in the chromatograms. A Hitrap Q HP column was used to fractionate different vanadium compounds in rat serum.

Arsenic speciation in chinese seaweeds using HPLC-ICP-MS and HPLC-ES-MS.

Three common Chinese edible seaweeds, one brown (Laminaria japonica) and two red (Porphyra crispata and Eucheuma denticulatum), were examined for their total arsenic content. The As species were extracted with yields of 76.4, 69.8 and 25.0%, respectively. Anion-exchange and cation-exchange high-performance liquid chromatography (HPLC) in combination with inductively coupled plasma mass spectrometry (ICP-MS) were used for the separation of the different arsenic species in two of the three seaweed extracts (Laminaria and Porphyra). The main arsenic species in the algal extracts are arseno sugars, although it has been shown that the Laminaria seaweed contains significant amounts of dimethylarsinic acid (DMA). HPLC was coupled with electrospray mass spectrometry (ES-MS) for structural confirmation of the arsenic species. The mass spectrometer settings for the arseno sugars were optimised using standards. The conclusions drawn on the basis of HPLC-ICP-MS were confirmed by the HPLC-ES-MS data. The HPLC-ES-MS method is capable of determining both arseno sugars and DMA in the seaweeds. The unknown compounds seen in the HPLC-ICP-MS chromatogram of Laminaria could not be ascribed to trimethylarsenic oxide or tetramethylarsonium ion.

Fractionation of vanadium in urine of Wistar rats as a function of time after intraperitoneal injection.

[(48)V]Vanadium was intraperitoneally injected into Wistar rats. Urine and feces were collected at regular intervals (n=19) between 1 and 144 h after injection. In case of urine, maximal excretion (V activity/ml urine) of vanadium was seen 3 h after injection. In case of feces, a maximum appeared 32 h after injection. Urine samples were fractionated on two types of gel filtration column (Superose 12 HR 10/30 and Superdex Peptide 10/30). We found that vanadium in urine exists as both high (protein-bound) and low molecular mass species and that the partition about these forms depends on the time elapsed after injection. After 1 h, respectively, four (one high molecular and three low molecular mass species) and five (one high molecular and four low molecular mass species) vanadium peaks were present in the chromatograms of the Superose 12 and the Superdex Peptide columns. Then 3 h after injection, a different high molecular species showed up in the chromatograms, while the first high molecular and some low molecular mass species disappeared. Vanadium in urine after 8 h occurred as one high (slightly different from the high molecular complex after 3 h) and one low molecular mass complex. However, after 48 h the pattern changed again and vanadium in urine was excreted largely as one low molecular mass species, presumably one of the species that also occurred 1 h after injection but was not present in the period 6-24 h.

The role of trace elements in uraemic toxicity.

Although most research on uraemic toxicity has focused on the retention or removal of organic solutes, subtle changes in the concentration of inorganic compounds are also of importance because these compounds may have significant clinical consequences. Potential clinical implications include increased risk of cancer, cardiovascular disease, immune deficiency, anaemia, renal function impairment and bone disease. In uraemic patients, the most important factor affecting trace element concentration is the degree of renal failure and modality of renal replacement therapy. Accumulation of trace elements in haemodialysis patients has resulted from dialysate contaminated with aluminium and strontium. Several trace elements have been implicated in the decline of renal function. These include arsenic, cadmium, copper, germanium, lead and mercury. In uraemic patients, aluminium, cadmium, chromium, lanthanum, strontium and zinc have been shown to accumulate in bone. In addition to substantial evidence linking aluminium to renal osteodystrophy, studies have also implicated cadmium, iron and strontium in bone disease. Studies using a rat model of chronic renal failure have demonstrated an association between lanthanum accumulation and mineralization defects characteristic of osteomalacia. Investigations of arsenic accumulation in animal models have demonstrated that speciation of trace elements potentially may alter toxicities of trace elements accumulated in uraemic patients. Conversely, the presence of uraemic toxins may also alter the uptake and toxicity of certain trace elements. Although research in uraemic patients has focused primarily on total concentrations of trace elements, the evolution of both inorganic and organic species should be considered separately.