Synthesis of [75Se]trimethylselenonium iodide from [75Se]selenocystine

N-Acetylglucosamine-6-sulfate sulfatase activity was assayed by incubation of the radiolabeled monosaccharide N-acetylglucosamine [1-14C]6-sulfate (GlcNAc6S) with homogenates of leukocytes and cultured skin fibroblasts and concentrates of urine derived from normal individuals, patients affected with N-acetylglucosamine-6-sulfate sulfatase deficiency (Sanfilippo D syndrome, mucopolysaccharidosis type IIID), and patients affected with other mucopolysaccharidoses. The assay clearly distinguished affected homozygotes from normal controls and other mucopolysaccharidosis types. The level of enzymatic activity toward GlcNAc6S was compared with that toward a sulfated disaccharide and a sulfated trisaccharide prepared from heparin. The disaccharide was desulfated at the same rate as the monosaccharide and the trisaccharide at 30 times that of the monosaccharide. Sulfatase activity toward glucose 6-sulfate and N-acetylmannosamine 6-sulfate was not detected. Sulfatase activity in fibroblast homogenates with GlcNAc6S exhibited a pH optimum at pH 6.5, an apparent Km of 330 mumol/liter, and inhibition by both sulfate and phosphate ions. The use of radiolabeled GlcNAc6S substrate for the assay of N-acetylglucosamine-6-sulfate sulfatase in leukocytes and skin fibroblasts for the routine enzymatic detection of the Sanfilippo D syndrome is recommended.     

[75Se]selenite and [75Se]selenate fluctuations during the development of murine hepatoma

The distribution of selenate and selenite selenium in C57L/J mice during the progression of BW7756 murine hepatoma was investigated using intra-ocular injection with the oxyanions labeled with ⁷⁵Se radioisotope. Comparison is made with the normal distribution of selenium studied by the RIXRF method. The trace elemental profiles, TEP, for the two oxidation states are compared in healthy and disease states. It has been found that the presence of tumor significantly changes the level of tracer in various uninvolved organs. These changes are prominent in the early growth phase of the tumor. The two oxidation states show differences in the TEP for kidney, spleen, stomach and testes.     

Assessment of platelet production with 75Se selenomethionine

Incorporation of intravenously injected ⁷⁵Se selenomethionine into platelets has been found to vary with alterations in the rate of platelet production. It appears to label newly produced platelets during their formation in megakaryocytes and provides a method by which thrombopoiesis may be studied in vivo. This technique may be applicable to clinical studies of disordered platelet production.     

Uptake of75Se in cataractous rat lens proteins

The purpose of the present study was to measure the pattern of uptake of⁷⁵Se into proteins in normal rat lenses and into the proteins of lenses with selenite-induced cataract. Ten-day-old suckling rats received a single injection of⁷⁵Se with or without a cataractous dose of cold carrier sodium selenite. Four days after injection, the proteins from excised lenses were counted for⁷⁵Se radioactivity and subjected to gel permeation chromatography, amino acid analyses, and mass spectrometry. All three soluble crystallin lens proteins took up⁷⁵Se in both normal and cataractous lenses. However, cataractous lenses did not take up⁷⁵Se into a soluble protein in which major quantities of⁷⁵Se were taken up in normal rats. Futhermore,⁷⁵Se in the gamma-crystallins was associated with an unusual acidic amino acid. It was concluded that selenium metabolism by lens proteins may be unusual compared to other soft tissues.     

Study of mammalian selenocysteyl-tRNA synthesis with [75Se]HSe

The mechanisms of the synthesis of mammalian selenocysteyl-(Scy)-tRNA were studied using [75SE]H2Se. H2Se was prepared from [75Se]selenite, glutathione, NADPH and glutathione reductase, and was purified by chromatography. It was confirmed that this H2Se was a Se donor in the reaction of the synthesis of Scy-tRNA. [75Se]Scy, liberated from aminoacyl-tRNA, was analyzed by TLC on silica gel an subsequent autoradiography. The activity of Scy-tRNA synthesis was found in the supernatant at 105,000 x g of the murine liver extract, but not in the precipitate. The supernatant was chromatographed on DEAE-cellulose, and the activity was eluted at a concentration of 0.17 M KCl. This position is at the front shoulder of the peak of seryl-tRNA synthetase which was eluted at 0.20 M KCl. Major serine tRNA(IGA) is not a substrate on which to synthesize Scy-tRNA, but natural opal suppressor serine tRNA is. On a chromatographic pattern of a Scy-tRNA preparation on Sephacryl S-200, the radioactivity of 75Se was eluted at the tRNA peak. This showed that Scy bound to tRNA. The active protein fraction from DEAE-cellulose did not contain tRNA kinase, therefore Scy-tRNA must be directly synthesized from seryl-tRNA, not through phosphoseryl-tRNA. This mechanism is similar to that seen in Escherichia coli [1991, J. Biol. Chem. 266, 6324].     

Incorporation of Radioactive Seleno-(75Se)-Methionine into Mumps Virus

Mumps virus was grown in embryonated chicken eggs in the presence of radioactive seleno-(⁷⁵Se)-methionine. Virus in the allantoic and amniotic fluids was concentrated in a sucrose density gradient, and a peak of viral material coincided with a significant peak of ⁷⁵Se-radioactivity. The radioactivity was acid-insoluble and remained associated with the virus after purification by erythrocyte adsorption and elution and centrifugation on a second sucrose density gradient. After amino-acid hydrolysis of the radioactive virus, only ⁷⁵Se-methionine was recovered by chromatographic analysis. These results demonstrate that the radioactive ⁷⁵Se-methionine was incorporated into protein of infectious mumps virus.     

A comparison of the uptake of [75Se]selenite, [75Se]selenomethionine and [35S]methionine by tissues of ewes and lambs.

The fate of selenium, given as Na2(75)SeO3, or [75Se]selenomethionine, and of [35S]methionine administered intravenously to ewes and lambs, has been examined. The main intention was to follow the incorporation of selenium into protein in a number of tissues, including liver and kidney, and to measure the extent of that incorporation of selenoamino acid, particularly with respect to the administration of selenite. The ewes chosen were lactating ewes with lambs at foot, and the lambs were animals which had been weaned on to fodder low in selenium and were recovering from white muscle disease with selenium therapy. These two experimental situations were chosen as they offered conditions under which selenium incorporation might be considered to be maximal. Entry of isotope into milk was rapid and was greater when 75Se was given as the selenoamino acid than as selenite. In both ewes and lambs greater amounts of activity, derived from selenite, were bound to plasma proteins than to the proteins of milk. This was particularly evident in samples taken some hours after administration. This ability of the plasma to bind selenium was demonstrated by alkaline dialysis. Small, though significant amounts of selenium, derived from Na2(75)SeO3, were incorporated as selenoamino acids into the proteins of liver, kidney and pancreas, as well as into the proteins of milk and plasma. In ewes, both selenomethionine and selenocystine were identified chromatographically in enzyme digests of defatted liver and kidney. Some differences occurred in the distribution of labelled compounds in organs from lactating ewes and recovering lambs. The incorporation of selenium into protein is discussed briefly in relation to the recent findings of an association between selenium and the enzyme glutathione peroxidase.     

An external-sample liquid scintillation counting for 75Se and its application to selenoprotein detection

An external-sample liquid scintillation (LS) counting for the gamma emitter ⁷⁵Se has been developed. An expressly designed well-type LS vial and a 2,5-diphenyoxazole-1,4-bis(5-phenyl-2-oxazoyl)-benzene-xylene solution containing 35% tertrabutylzinn allow ⁷⁵Se to be counted in a standard LS counter with counting efficiency up to 43.2%, much higher than that of conventional LS counting method. This external sample LS has a good count rate linearity and exhibits low background count rates. After in vivo labeling with [⁷⁵Se]selenite, ⁷⁵Se distributions and the Se-containing proteins present in tissues of male rat were investigated by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, external-sample LS and γ-detector. Eight Se-containing proteins or protein subunits were detected to be Se-containing proteins or protein subunits in arterial wall, and their apparent molecular masses (Mr) were 76.4, 67.0, 57.4, 30.3, 25.4, 22.7, 21.7, and 15.1 kDa, respectively. In addition, eight ⁷⁵Se-labeled proteins (Mr: 66.8, 57.0, 43.1, 30.0, 24.8, 19.8, 18.0, and 14.8 kDa) were found in brain homogenates, and nine ⁷⁵Se-labeled proteins (Mr: 117.0, 78.0, 66.6, 57.2, 43.0, 38.1, 25.0, 20.1, and 18.0 kDa) were detected in testis homogenates. Some of them should be new biologically important selenoproteins that have not been identified so far.     

Bioavailability of selenium from meat and broccoli as determined by retention and distribution of 75Se

The concentration of selenium (Se), an essential nutrient, is variable in foods, depending, in part, on how and where foods are produced; some foods accumulate substantial amounts of Se when produced on high-Se soils. The chemical form of Se also differs among foods. Broccoli is a Se-accumulating plant that contains many methylated forms of Se, and Se bioavailability from broccoli has been reported to be low. Red meats such as pork or beef could accumulate Se when the animal is fed high-Se diets, and Se from such meats has been reported to be highly bioavailable for selenoprotein synthesis. In a further attempt to characterize the utilization of Se from broccoli and meats such as pork or beef, we have fed rats diets adequate (0.1 μg Se/g diet) in Se or high in Se (1.5 μg S/g diet), with the Se source being either high-Se broccoli or beef. Rats were then given test meals of broccoli or pork intrinsically labeled with ⁷⁵Se. When dietary Se was nutritionally adequate (0.1 μg/g diet), more ⁷⁵Se from pork than broccoli was retained in tissues; however, there were no significant differences in whole-body retention when dietary Se was high (1.5 μg/g diet). A significantly greater percentage of ⁷⁵Se from broccoli than pork was excreted in the urine and dietary Se did not affect urinary excretion of broccoli ⁷⁵Se, but the amount excreted from pork varied directly with dietary Se intake. Radiolabeled ⁷⁵Se derived from pork effectively labeled selenoproteins in all tissues examined, but ⁷⁵Se from broccoli was undetectable in selenoproteins. These differences in retention and distribution of Se from broccoli or pork are consistent with reported differences in bioavailability of Se from beef and broccoli. They also suggest that there are fewer differences in bioavailability when Se is consumed in supranutritional amounts.     

Use of a simple gamma flow cell to monitor chromatography column effluents containing 75Se.

Scintillation flow cells provide a convenient means of monitoring column effluents for radioactively labeled compounds. The use of flow cells to monitor effluents containing ³H, ¹⁴C, and other β-emitting isotopes is well established (1) and such cells are readily available as accessories for use with liquid scintillation counters. β-Flow cells have occasionally been used to monitor γ emitters [e.g., Martin et al. (2)] but the counting efficiency is extremely low even for weak γ emitters such as ⁷⁵Se, due to the limited mass of scintillant available to absorb the radiation.Spencer et al. (3) described a simple γ flow cell consisting of a coiled polyethylene tube inserted in to the well crystal of a γ counter, but no information on counting efficiency was given. Although spiral plastic γ cells have been manufactured commercially (e.g., Nuclear Enterprises Ltd., NE702¹) they are not offered as a standard accessory for most common makes of γ counter and do not appear to be readily available from commercial sources.This communication describes a simple, low cost γ flow cell constructed for use in a study on selenium transport in higher plants, which involved the separation of ⁷⁵Se-labeled constituents in a large number of samples of xylem sap.     

A conjugate counting method to determine [75Se]SeHCAT retention in the human body

To evaluate the functional integrity of the distal part of the ileum the retention of a γ-labelled bile acid (SeHCAT) in the human body can be measured with a detector. Due to the lack of a whole body counter at our institution a two detector system was designed to measure SeHCAT retention and an evaluation of such a system has been made. The detectors are positioned on either side of a patient lying supine on a hospital trolley. The trolley is stepped forward in 100 mm steps, to determine the SeHCAT activity in the patient. With these counts the location of the SeHCAT activity and total activity present in the body can be determined. A water rilled phantom and a phantom consisting of nine 1-L saline bags with ⁷⁵Se activity placed in them was used to determine system performance. Four patients with no history of bowel disease were compared with published data for normals. Results showed that the system performed satisfactorily, and accurate quantitative measurements could be made, showing that this inexpensive system could be used where a whole body counter is not available.     

Selenium in the anterior pituitary of the rat after a single injection of75Se sodium selenite

In order to investigate the selenite metabolism in the anterior pituitary and compare it with other endocrine organs, rats were injected intraperitoneally with⁷⁵Se sodium selenite (5 mg/kg). The rats were whole body counted shortly after injection and recounted just before sacrifice, which was performed 2, 24, 48 h, and 4, 10, 20, 30, 40, 60, and 80 d after injection. Besides the anterior pituitary, the selenium content was also estimated in the thyroid gland, testis, adrenals, liver, kidney, and blood. The maximum selenium content was observed in all organs 2 h after injection, at which time the anterior pituitary contained 2.9 μg/g wet wt, compared to 13.5 μ/g wet wt in liver and .6 μg/mg wet wt in testis. The excretion of selenite from the anterior pituitary resembled that seen in most other organs investigated, i.e., an initial rapid excretion and a slower secondary phase resembling a first order reaction. Practically all selenium was excreted by 60 d after injection.