Distribution and metabolism of selenite and selenomethionine in the Japanese quail.

Compared to the many studies on the physiological and toxicological effects of selenium (Se) in mammals, avian Se metabolism is still an unexplored topic. Some birds are useful as poultry for human nutrition. Moreover, birds belong to higher trophic levels in the biosphere and thus may play an important role in Se circulation in the ecosystem in the same way as mammals do. In this study, we analyzed the distribution and metabolism of Se in an experimental bird, the Japanese quail, which was fed drinking water containing sodium selenite or selenomethionine (SeMet). The highest concentration of Se was detected in the pancreas, followed by down feathers, liver, and kidneys. SeMet was more efficiently incorporated into the quail than selenite. The specific and preferable distribution of Se to the high molecular weight fraction in the serum of the quail was observed only in the SeMet-ingestion group. As in mammals, selenosugar and trimethylselenonium were the major metabolites in quail excreta. Three unknown Se metabolites were detected by HPLC-ICP-MS. Although part of the metabolic pathway of Se in the Japanese quail fed selenite and SeMet was the same as that observed in mammals, the bird also showed certain avian-specific metabolic process for Se.

Human urinary excretion and metabolism of (82)Se-enriched selenite and selenate determined by LC-ICP-MS.

Urinary excretion of selenium after ingestion of isotope labeled selenite and selenate was studied in seven healthy volunteers, 4 men and 3 women (age 28-50 years). An aqueous solution containing 330 µL (82)Se-selenate (corresponding to 74.3 µg (82)Se) was given orally and urine samples were subsequently collected during the following 24 hours. The scheme was repeated four weeks later with a 280 µL (82)Se-selenite solution (corresponding to 74.4 µg (82)Se). The amount of total Se in the urine samples was determined by inductively coupled mass spectrometry. The mean total urinary excretion of (82)Se following (82)Se-selenate administration was 33.7% (range 15.6-42.5%) while the mean total excretion of (82)Se after (82)Se-selenite administration was 3.2% (range 2.8-3.9%) of the ingested amount. LC-ICPMS analysis of the urine samples showed that the majority of the selenium excreted after selenate ingestion was unchanged selenate for 6 of the individuals while one individual had metabolized a fraction (approx. 20%) of the selenate to selenosugar. Ingestion of 10 times larger doses of selenite in two individuals resulted in 13-23% excretion primarily excreted as selenosugar. These results show that the human metabolic pathways of selenite and selenate are different and indicate that not all selenate, although well absorbed, may be available for the beneficial health effects.

Simultaneous analysis of mercury and selenium species including chiral forms of selenomethionine in human urine and serum by HPLC column-switching coupled to ICP-MS.

The simultaneous speciation of elements is of great concern, especially in the study of the interactions of species in living organisms. Here we report a method based on the coupling of HPLC-ICP-MS that is capable of separating and analyzing different selenium and mercury species (Se-methylselenocysteine, selenite, selenate, L-selenomethionine, D-selenomethionine, methylmercury and inorganic mercury). The proposed method uses two different mobile phases that are suitable for selenium and mercury speciation and leads to a successful determination of all the species in less than 27 min with good efficiency and resolution. The method was efficiently applied for simultaneous speciation of mercury and selenium in urine and in serum, the latter from umbilical cord samples. Selenocystine has been successfully identified in the former sample. Detection limits obtained were between 0.30 and 2.46 ng. Recovery studies of samples spiked with all species were performed to check the reliability of the method, and satisfactory recoveries (93-110%) were obtained in all cases. The relative standard deviations (RSDs) for species with ten replicate determinations of 80 µg L(-1) were between 4.5 and 9.2%. The proposed method offers a deeper insight into selenium and mercury interactions in the human body.

Comparison of tetraethylborate and tetraphenylborate for selenite determination in human urine by gas chromatography mass spectrometry, after headspace solid phase microextraction.

Two different derivatizing reagents were tested for the development of a fast and sensitive method for the determination of selenites (Se(IV)) in human urine. The reagents were sodium tetraethylborate (NaBEt(4)) and tetraphenylborate (NaBPh(4)), respectively, and the procedure is based on in situ derivatization of selenites in aqueous medium. Selenite ions are converted to diethylselenide (DESe) or diphenylselenide (DPhSe) and subsequently collected from the headspace by solid phase microextraction using a silica fiber coated with polydimethylsiloxane (HS-SPME). Finally, they are quantitated by GC/MS in SIM mode. Ethylation over phenylation was proved preferable for the headspace extraction because of the higher volatility of the diethyl-derivative of selenites. The optimization of the HS-SPME conditions was performed both in aqueous and urinary solutions. Under the optimum conditions for HS-SPME, the gas chromatographic conditions were also optimized. Between the two alkylation reagents tetraethylborate was proved more efficient and the quantitation was satisfactory. Aqueous certified reference materials were analyzed to evaluate the accuracy of the method. The precision of the method was 4.2% and the calculated detection limit was 0.05microgL(-1) for human urine.

Headspace and direct immersion solid phase microextraction procedures for selenite determination in urine, saliva and milk by gas chromatography mass spectrometry.

Two solid phase microextraction modes were investigated and compared for their performance on the determination of selenites in various biological liquids like human urine and saliva and various types of milk. Using sodium tetraethylborate (NaBEt(4)) as ethylating reagent, selenites are converted in situ to volatile diethylselenides (DESe) in aqueous medium. The derivative is collected in situ by solid phase microextraction (SPME) using a silica fiber coated with poly(dimethylsiloxane) (PDMS) either from the headspace (HS-SPME) or directly from the liquid phase (LP-SPME) and finally determined by capillary GC/MS. Under optimum conditions of SPME, the GC separation was also optimized. Between the two examined microextraction techniques, direct immersion of the PDMS fiber in the liquid phase was proved less satisfactory. In contrast, the headspace procedure appears to be more efficient. The quantification of selenites was achieved in SIM mode with good analytical performance. A non-fat milk powder certified reference material was analyzed to evaluate the accuracy of the method. The overall precision of the method was ranged between 6.2% and 9.7%. Detection limits achieved were 0.05microgL(-1) for human urine, 0.08microgL(-1) for saliva and 0.03-0.06microgL(-1) in various milk matrices.

Metabolism of 76Se-methylselenocysteine compared with that of 77Se-selenomethionine and 82Se-selenite.

Se-Methylated selenoamino acids, Se-methylselenocysteine (MeSeCys) and selenomethionine (SeMet), are chemically inert storage forms of selenium in selenium-accumulators, and a nutritional and supplemental source. The metabolic pathway for MeSeCys was precisely traced by referring to those for SeMet and selenite by applying a new tracer method involving multiple homo-elemental stable isotopes. Male Wistar rats were depleted of endogenous natural abundance selenium with a single (80)Se-enriched isotope, and then (76)Se-MeSeCys, (77)Se-SeMet and (82)Se-selenite were orally administered simultaneously at 25 microg Se/kg body weight each. Organs and body fluids were obtained at 3, 6, 9 and 12 h, and 1 and 2 days later, and subjected to speciation analysis. The main characteristics of the metabolism were as follows; MeSeCys was incorporated into selenoprotein P slightly more than or at a comparable level to that of SeMet but less than that of selenite. MeSeCys and SeMet but not selenite was taken up by organs in their intact forms. MeSeCys and SeMet were delivered specifically to the pancreas and present in a form bound to an identical or similar protein. Trimethylselenonium (TMSe) was only produced from MeSeCys, i.e., not from SeMet or selenite, in the kidneys. Both selenosugars A and B of MeSeCys, SeMet and selenite origin were detected in the liver but only selenosugar B in the kidneys. These results suggest that MeSeCys can be a similar or better selenium source than SeMet, and supplies methylselenol much more efficiently in organs than SeMet and selenite. TMSe was produced much efficiently from MeSeCys than from SeMet and selenite, suggesting a role of methylselenol through the beta-lyase reaction in the metabolism of Se-methylated selenoamino acids.

Simultaneous tracing of 76Se-selenite and 77Se-selenomethionine by absolute labeling and speciation.

Nutritional selenocompounds are transformed into the assumed common intermediate selenide, which is utilized for the synthesis of selenoenzymes or transformed into methylated metabolites for excretion. Hence, selenocompound metabolites can be traced only with labeled selenium. Here we applied a new tracer method for the metallomics of biometals using simultaneous speciation of each metallome labeled with different homo-elemental isotopes to metabolism and availability of selenium. Rats were depleted of endogenous natural abundance selenium by feeding a single selenium stable isotope ((82)Se-selenite) and then administered (76)Se-selenite and (77)Se-selenomethionine ((77)Se-SeMet)simultaneously. Biological samples were subjected to quantification and speciation analysis by HPLC-ICPMS. Metabolites of the labeled (76)Se and (77)Se and interaction with endogenous selenium were traced and examined without interference from the corresponding endogenous natural abundance isotopes. Differences in the distribution and metabolism among organs and between the two nutritional selenocompounds were compared under exactly identical biological and analytical conditions: (1) selenite was distributed more efficiently than SeMet in organs and body fluids except the pancreas. (2) SeMet was taken up by organs in its intact form. (3) Selenium of SeMet origin was distributed selectively in the pancreas and mostly bound to a protein together with intact SeMet. (4) Selenosugars A and B but not trimethylselenonium (TMSe) were detected in the liver. (5) Selenosugar B and TMSe were detected in the kidneys.

Availability and metabolism of 77Se-methylseleninic acid compared simultaneously with those of three related selenocompounds.

Nutritional selenocompounds are considered to be transformed into the common intermediate selenide for utilization as selenoenzymes and/or for excretion as selenosugar and trimethylselenonium (TMSe). Therefore, selenocompounds can only be traced with a labeled selenium atom. Methylseleninic (MSA(IV)) has been proposed to be a third nutritional selenium source, the other two being inorganic selenocompounds and organic selenoamino acids, and to be a proximate selenochemical for producing the assumed biologically active form methylselenol. Here we applied a new tracer method to compare the availability and metabolism of MSA(IV) with those of three related selenocompounds under exactly identical host and tracing conditions. (82)Se-Selenite, (78)Se-selenate, (77)Se-MSA(IV) and (76)Se-methylselenonic acids (MSA(VI)) were simultaneously administered orally, each at the dose of 25 microg Se/kg body weight, to rats that had been depleted of endogenous natural abundance selenium with a single stable isotope ((80)Se). Time-related changes in the concentrations and/or distributions of the four labeled isotopes in the serum, liver, kidney, pancreas, lung and urine were determined simultaneously by inductively coupled argon plasma mass spectrometry (ICP MS) and/or HPLC-ICP MS. The availability with different isotope ratios was in the decreasing order of selenate>selenite=MSA(IV)>MSA(VI). Although selenate and MSA(VI) were distributed in organs and urine partly in their intact forms, MSA(IV) and selenite were not detected in the intact forms at all. MSA(IV) and MSA(VI) but not selenite or selenate produced TMSe in organs other than the liver, suggesting the transformation of MSA(IV) into methylselenol, and then either into selenide for the synthesis of selenoproteins and selenosugar or directly into TMSe. Thus, selenosugar and TMSe were produced widely in the organs. However, TMSe was not detected in the liver. The organ- and selenium source-specific production of TMSe was discussed as to the differences in selenium sources, and demethylation and methylation activity.

Distribution and reuse of 76Se-selenosugar in selenium-deficient rats.

Nutritional selenium compounds are transformed to the common intermediate selenide and then utilized for selenoprotein synthesis or excreted in urine mostly as 1beta-methylseleno-N-acetyl-Dd-galactosamine (selenosugar). Since the biological significance of selenosugar formation is unknown, we investigated their role in the formation of selenoenzymes in selenium deficiency. Rats were depleted of endogenous natural abundance selenium with a single stable isotope ((82)Se) and then made Se-deficient. (76)Se-Selenosugar was administered intravenously to the rats and their urine, serum, liver, kidneys and testes were subjected to speciation analysis with HPLC inductively coupled argon plasma mass spectrometry. Most (76)Se was recovered in its intact form (approximately 80% of dose) in urine within 1 h. Speciation analysis revealed that residual endogenous natural abundance selenium estimated by (77)Se and (78)Se was negligible and distinct distributions of the labeled (76)Se were detected in the body fluids and organs without interference from the endogenous natural abundance stable isotope. Namely, intact (76)Se-selenosugar was distributed to organs after the injection, and (76)Se was used for selenoprotein synthesis. Oxidation to methylseleninic acid and/or hydrolysis of the selenoacetal group to methylselenol were proposed to the transformation of selenosugar for the reuse. Effective use of an enriched stable isotope as an absolute label in hosts depleted of natural abundance isotopes was discussed for application in tracer experiments.

Selenium speciation by high-performance anion-exchange chromatography-post-column UV irradiation coupled with atomic fluorescence spectrometry.

A technique for the speciation of selenomethylcysteine (SeMeCys), selenocystine (SeCys), selenite [Se(IV)] and selenomethionine (SeMet) was established in this paper using high-performance anion-exchange chromatography coupled with atomic fluorescence spectrometry (HPAEC-AFS). Analytes were separated on an AminoPac PA10 column and then digested by on-line ultraviolet (UV) irradiation, which destroyed organic compound structure. Hydride generation was used as an available sample introduction technique for atomic fluorescence detection. The detection limits of four compounds were 1-5 microg/L (250 microL injection, 10 times of the baseline noise). The relative standard deviations (RSDs), calculated from seven consecutive injections of 100 microg/L standard mixtures, were from 2 to 4%. Selenious yeast tablet, which had been proposed as selenium supplement, and human urine collected from a volunteer were analyzed. Good spiked recoveries from 86 to 103% were obtained.

Selenium metabolites in human urine after ingestion of selenite, L-selenomethionine, or DL-selenomethionine: a quantitative case study by HPLC/ICPMS.

To obtain quantitative information on human metabolism of selenium, we have performed selenium speciation analysis by HPLC/ICPMS on samples of human urine from one volunteer over a 48-hour period after ingestion of selenium (1.0 mg) as sodium selenite, L-selenomethionine, or DL-selenomethionine. The three separate experiments were performed in duplicate. Normal background urine from the volunteer contained total selenium concentrations of 8-30 microg Se/L (n=22) but, depending on the chromatographic conditions, only about 30-70% could be quantified by HPLC/ICPMS. The major species in background urine were two selenosugars, namely methyl-2-acetamido-2-deoxy-1-seleno-beta-D-galactopyranoside (selenosugar 1) and its deacylated analog methyl-2-amino-2-deoxy-1-seleno-beta-D-galactopyranoside (selenosugar 3). Selenium was rapidly excreted after ingestion of the selenium compounds: the peak concentrations (approximately 250-400 microg Se/L, normalized concentrations) were recorded within 5-9 hours, and concentrations had returned to close to background levels within 48 hours, by which time 25-40% of the ingested selenium, depending on the species ingested, had been accounted for in the urine. In all experiments, the major metabolite was selenosugar 1, constituting either approximately 80% of the total selenium excreted over the first 24 hours after ingestion of selenite or L-selenomethionine or approximately 65% after ingestion of DL-selenomethionine. Selenite was not present at significant levels (<1 microg Se/L) in any of the samples; selenomethionine was present in only trace amounts (approximately 1 microg/L, equivalent to less than 0.5% of the total Se) following ingestion of L-selenomethionine, but it constituted about 20% of the excreted selenium (first 24 hours) after ingestion of DL-selenomethionine, presumably because the D form was not efficiently metabolized. Trimethylselenonium ion, a commonly reported urine metabolite, could not be detected (<1 microg/L) in the urine samples after ingestion of selenite or selenomethionine. Cytotoxicity studies on selenosugar 1 and its glucosamine isomer (selenosugar 2, methyl-2-acetamido-2-deoxy-1-seleno-beta-D-glucosopyranoside) were performed with HepG2 cells derived from human hepatocarcinoma, and these showed that both compounds had low toxicity (about 1000-fold less toxic than sodium selenite). The results support earlier studies showing that selenosugar 1 is the major urinary metabolite after increased selenium intake, and they suggest that previously accepted pathways for human metabolism of selenium involving trimethylselenonium ion as the excretionary end product may need to be re-evaluated.

Determination of selenium compounds in urine by high-performance liquid chromatography--inductively coupled plasma mass spectrometry.

Selenium species, selenite, selenate, selenomethionine (Semet), seneloethionine (Seet) and trimethylselenonium ion (TmSe) were separated in aqueous solution using a gel-permeation (polyvinyl alcohol-based resin) GS-220 column by eluting with 25 mM tetramethylammonium hydroxide and 25 mM malonic acid at pH 7.9. The GS-220 column coupled with inductively coupled plasma mass spectrometry was used for the separation, identification, and quantification of selenium compounds present in certified reference material (CRM) No. 18 human urine from the National Institute for Environmental Studies in Japan (NIES). Spiking of the authentic standard to the urine and use of a silica-based LC-SCX cation-exchange column validated the peak of selenium compounds. High concentrations of chloride and bromide in the urine eluted from the GS-220 column formed molecular ions 40Ar37Cl+ and 81Br1H+ in the plasma, and these molecular ions created additional peaks in the chromatograms when 77Se and 82Se isotopes were monitored respectively. Thus, both the isotopes were selected concurrently for signal monitoring to eliminate the interfering signals. On the LC-SCX column, chloride and bromide were eluted with selenate and complicated its determination, but the peak of TmSe was baseline separated from rest of the Se compounds. Two unknown Se compounds were detected in both the columns. An additional Se compound having the same retention time as that of Semet was detected on the LC-SCX column. Peaks of selenite, selenate, TmSe and unknown selenium compounds in the urine were baseline separated on the GS-220 column, and were free from interferences. Therefore, the GS-220 column was used for the determination of selenium compounds in NIES CRM No. 18. Unknown Se compounds were the predominant selenium species followed by selenite, TmSe and selenate. The estimated value of TmSe as Se, by the standard additions method using the GS-220 column, was 3.42 +/- 0.17 microg l(-1) and was in good agreement with the LC-SCX value [3.38 +/- 0.21 (n=5) microg l(-1)].

Effects of arsenic-, platinum-, and gold-containing drugs on the disposition of exogenous selenium in rats.

Having found that the electrophilic model compound sulfobromophthalein markedly altered the fate of exogenous selenium in the body by reacting in vivo with nucleophilic selenium metabolites, the effects of metal-containing drugs with expected selenium reactivity were tested on biliary, urinary, and pulmonary excretion. Tissue distribution of selenium in selenite-injected rats was also examined. Coadministration with [(75)Se]selenite (10 micromol/kg, iv) of the trypanosomicid arsenicals (100 micromol/kg, iv) trimelarsan (TMA) or melarsoprol (MAP), the antitumor cisplatin (25 micromol/kg, iv), or the antirheumatic gold sodium thiomalate (25 or 50 micromol/kg, iv) significantly altered the disposition of (75)Se, whereas carboplatin (100 micromol/kg, iv) did not produce such an effect. The most dramatic alterations included the approximately 20-fold increase in the biliary excretion rate of selenium in response to TMA and MAP, the almost complete cessation of the exhalation of selenium as dimethyl selenide after administration of the arsenic- and gold-containing drugs, and the manifold accumulation of selenium in the blood plasma following gold injection. Direct chemical reaction of the drugs with nucleophilic selenite metabolites in the body may underlie these alterations. The tight coordination in time and extent observed between the biliary excretion of arsenic and selenium in rats receiving either of the arsenicals and selenite supports this hypothesis. However, attempts to detect selenium-containing biliary metabolites of TMA and MAP have failed, possibly owing to their instability. In summary, the arsenic-, platinum- and gold-containing drugs significantly influence the fate of exogenous selenium, whereby they may adversely affect the availability of this essential element for synthesis of selenoenzymes. Furthermore, the capability of TMA and MAP to enhance the biliary and total excretion of selenium renders these drugs significant candidates for antidotes in selenium intoxication.

Effects of dietary selenium species on Se concentrations in hair, blood, and urine.

The effects of the chemical species and concentration of selenium (Se) in diets on the concentrations of Se in hair, blood serum, red blood cells (RBCs), and urine were studied to gain an insight into the toxicological and nutritional significance of different chemical forms of Se. Male Wistar rats were fed an Se-deficient diet (Se, less than 0.03 microgram/g) for 3 weeks, and then an Se-adequate (Se, 0.2 microgram/g) or Se-excess diet (Se, 2.0 micrograms/g), including seleno-L-methionine (SeMet) or selenite for up to 12 weeks. Hair, blood, and urine specimens were obtained every two weeks, and the concentrations of Se and its distribution in serum and urine on a size-exclusion column were determined. The concentrations of Se in hair, serum, and urine attained constant levels 2 weeks after a change of in the dietary Se concentration irrespective of the chemical species, the levels being dependent on the chemical species and the concentration. Specifically, in hair and serum, selenite gave the lowest constant levels irrespective of the dose, while SeMet resulted in higher levels than selenite in a dose-dependent manner. The two major selenoproteins in serum exhibited comparable concentrations. On the other hand, in urine, the concentration of Se was dependent on the dose but not on the chemical species. The results could be explained by regulated metabolism of selenite, and both nonregulated and regulated aspects of the metabolism of SeMet.

Effects of dose on the methylation of selenium to monomethylselenol and trimethylselenonium ion in rats.

Mechanisms and metabolic significance in rats of methylation to the reduced form of selenium (Se), i.e., selenide (Se2-), were studied by dose- and time-related experiments with injection of selenite. Urinary Se-metabolites were determined by HPLC using an inductively coupled argon plasma-mass spectrometer as an in-line detector (HPLC/ICP-MS method). Although only monomethylselenon (MMSe) has been detected in urine of normal rats even in those fed a Se-excess diet, the three types of Se-metabolites - MMSe, trimethylselenonium ion (TMSe), and inorganic Se, were detected in urine of Wistar rats injected with selenite (0, 0.1, 0.3, 0.5 and 1.0 mg Se/kg body weight) into the tail vein. The amount of the three Se-metabolites was plotted against the total urinary Se concentration and shown to change dose- and time-dependently. The monomethylated metabolite, i.e., MMSe, increased in urine rapidly at first and was slowly followed by linear dose-dependent excretion of the trimethylated metabolite, TMSe. The new methylation pathway of MMSe leading to TMSe was assumed to be induced or activated when the dose of Se exceeds the limit of the normal capacity for monomethylation. Progressive methylation reactions were suggested to be regulated enzymatically.

Metabolites of sodium selenite and methylated selenium compounds administered at cancer chemoprevention levels in the rat.

1. The metabolism of orally-administered sodium selenite and five methylated selenium compounds was investigated in the female rat at dosages equivalent to those used in other studies for prevention of mammary cancer. Dimethyl selenide (DMSe) exhaled within 24 h following dosing was measured, along with inorganic and monomethylated (MMSe) forms of selenium plus trimethylselenonium ion (TMSe+) in urine. 2. MMSe was the dominant metabolite of selenite given at low levels (0.1 ppm in the diet), but excretion of DMSe and TMSe+ increased sharply when selenite dosage was increased to the chemopreventive range of 3 ppm dietary Se. When similar chemopreventive levels of mono-, di-, or trimethylated compounds were administered, the total quantity of methylated metabolites was greater than for selenite and the metabolite profile reflected the expected point of entry into the intermediary metabolism pathway; the major metabolites were MMSe from Se-methylselenocysteine, DMSe from selenobetaine methyl ester, and TMSe+ from selenobetaine. However, the profile of metabolites provided clear evidence that the methylated selenium compounds underwent demethylation, as shown by the excretion of inorganic and MMSe. Selenium administered as dimethyl selenoxide was almost completely excreted and about 90% of the dose was recovered as DMSe, indicating that reduction was the major pathway. For TMSe+, about 10% of the dose was excreted as DMSe and 84% as TMSe+. 3. A low, non-toxic level of sodium arsenite (5 ppm As in the diet) that is known to modify differentially the anticarcinogenic activity of selenite and methylated selenium compounds did not modify the excretion of the methylated selenium metabolites. 4. It is concluded that high anticarcinogenic activity is associated with extensive excretion of methylated Se excretory metabolites, but high output of such metabolites per se does not necessarily lead to anticarcinogenic activity. The whole animal has extensive capabilities for interconverting forms of selenium, and retains significant amounts in tissues, complicating the interpretation of Se metabolism and anticarcinogenic action. Further research is needed on the forms of selenium present in tissues.