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Interactions of gold with cytosolic selenium-containing proteins in rat kidney and liver.

Rats injected with aurothioglucose (ATG) for 5 days were subsequently injected with [75Se]selenious acid and killed after 3 days. Kidney and liver cytosols were chromatographed on Sephadex G-150. 75Se in kidney was associated with high molecular weight (HMW), 85,000 Mr, 26,000 Mr, and 10,000 Mr proteins and with a nonprotein fraction. The elution profile of liver cytosol was similar to that of kidney, but without a 26,000 Mr protein. ATG injection increased the association of 75Se with all fractions of kidney cytosol except the 85,000 Mr fractions, which contained Se-glutathione peroxidase (SeGSHPx) activity; 75Se in liver was increased only in HMW fractions. Unfractionated kidney cytosolic SeGSHPx activity was decreased 14% by ATG injection, but liver enzyme activity was not changed. However, Sephadex G-150 chromatography showed that total and specific activities, respectively, were decreased 28 and 23% in kidney and 25 and 16% in liver. Au coeluted with HMW and 10,000 Mr 73Se-containing kidney proteins; the latter contained 50% of the Au eluted from the column. DEAE Sephacel chromatography of the 10,000 Mr kidney protein showed that both Au and 75Se were tightly associated with metallothionein-like proteins. This study demonstrates the interaction of Au with rat liver and kidney 75Se-containing proteins.

Animals↗

75Selenium-labeled sheep plasma: the time course of changes in 75selenium distribution.

Sheep fed rations containing 0.1 ppm selenium were labeled by intravenous injection of radioactive sodium selenite or selenocystine. Gel filtration of serially collected plasma samples indicated that, with time, there was a transition from mercaptan sensitive to high mol wt mercaptan and protein solubilizer resistant selenoproteins. Radiolabeled plasma samples collected from selenite and selenocystine labeled sheep were dialyzed against buffer containing 2-mercaptoethanol or protein solubilizer. No difference in the stability between selenite- and selenocystine-labeled plasma could be detected.

Animals↗

Selenium-containing proteins of rat kidney and liver microsomes.

Selenium (Se)-containing proteins in microsomal fractions of rat kidney and liver were investigated after isotopic labeling of rats with [75Se]selenite. More than 85% of the 75Se in the solubilized microsomal extracts precipitated with protein after trichloroacetic acid treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), used to separate the labeled protein subunits in the solubilized microsomal extracts, revealed several 75Se-containing proteins in addition to glutathione peroxidase. 75Se-labeled subunits with molecular weights of 55, 30, 26, 22, 19, and 17 kDa were present in microsomal fractions of kidney and liver. The 75Se-labeled tryptic peptide of the 55 kDa subunit had the same Rf value on a 17% SDS-PAGE gel as the peptide from plasma selenoprotein P. A time-course study of the labeling of individual protein subunits in kidney and liver microsomes from Se-supplemented and Se-deficient rats showed that most of the 75Se was associated with the 55 kDa subunit 3 hr after injection. The amount of 75Se associated with this protein subunit decreased by 12 hr, with a concurrent increase in the labeling of lower molecular-weight subunits. The results support the hypothesis that there is a mechanism for transfer of Se from the 55 kDa subunit to other Se-containing proteins.

Animals↗

Uptake of selenotrisulfides of glutathione and cysteine by brush border membranes from rat intestines.

The uptake of selenodiglutathione and selenodicysteine was compared to that of selenite by brush border membrane vesicles (BBMV) prepared from rat intestinal tracts. It was found that it is critical to maintain a pH of 6.0 or below to prevent the spontaneous breakdown of these compounds. When conducted at pH 6.0, the uptake of selenodiglutathione and selenodicysteine was more than ten times faster than for selenite selenium. Ligated intestinal loop studies were conducted to determine if similar results would be obtained in vivo. In comparison to selenite, selenium absorption was enhanced 68% and the transfer to the body increased 2.4-fold when selenium as selenodiglutathione was placed in the ileum. The absorption of selenium as selenodicysteine was increased by 57% and the transfer doubled in comparison to selenite when placed in the ileum. Thus, the stimulated absorption of selenite by glutathione or cysteine appears to be through the formation of complexes with these compounds.

Animals↗

In vivo labelling of viral proteins with 75selenomethionine.

A method is described for in vivo labelling of viral proteins with a gamma ray emitting radionuclide. The amino acid methionine labelled with 75Se instead of 35S will be accepted by cells in culture for protein synthesis. A high specific labelling of viral proteins is achieved which allows rapid detection of virus or viral proteins in gradient centrifugation and PAGE as well as further biochemical or serological analysis of the proteins. As an example hog cholera virus (family Togaviridae) was labelled with [75Se]methionine and analyzed in sucrose and CsCl gradients and in PAGE. The results were similar to those obtained by labelling of proteins with [35S]methionine.

Centrifugation, Density Gradient↗

A comparison of the effects of dietary selenium on selenoprotein expression in rat brain and liver.

In studies with rodents, when dietary supplies of the essential nutrient Se are restricted, in most tissues there are parallel substantial losses of the element and the important antioxidant selenoenzyme glutathione peroxidase (GPx) for which it is a cofactor. In brain, however, there appears to be both a sequestration of Se and a conservation of GPx activity when dietary Se is limited. To further explore the relation between these phenomena, we have undertaken a comparison of the effects of diets low, normal and high in Se on GPx activity, and labeling of selenoproteins following short-term (72 h) in vivo exposure to 75Se, in subcellular fractions from rat brain and liver, the latter serving as a representative tissue which does not retain Se and is depleted of most GPx activity following dietary restriction. Brains and livers from animals on the three diets showed different patterns of response with respect to both GPx activity and retention of the 75Se dose. The low-Se diet (0.006 ppm) substantially reduced GPx activity in liver but not brain, while high levels (1 ppm) did not increase GPx in either tissue relative to a normal (0.1 ppm) intake. The 75Se was retained in brain homogenates and subcellular fractions to the greatest extent by rats on the restricted diet, while in liver, retention was greater in rats fed the normal supplement than in animals on either the low- or high-Se diets. Levels of non-protein-bound 75Se were higher in brain than liver and increased with dietary Se in both tissues. When proteins in brain and liver homogenates and subcellular fractions where separated by one-dimensional SDS-PAGE and exposed to X-ray film, the resulting autoradiograms revealed the existence of seven distinct selenoprotein bands in brain and eight in liver. Different patterns of selenoprotein expression were observed in subcellular fractions isolated from both tissues. Dependence of levels of individual selenoproteins on diet paralleled the effects on 75Se retention. Dietary influences on expression of protein bands tentatively identified as GPx were more pronounced in liver than brain. All of these observations provide further evidence of the unique nature of Se metabolism in brain.

Animals↗

Toxicity, metabolism and absorption of selenite by isolated rat hepatocytes.

The uptake of various levels of selenite by isolated rat hepatocytes was investigated. The LD50 value of selenite was about 500 microM. The activity of lactic dehydrogenase in the medium was correlated with cell viability as determined by trypan blue exclusion. After incubation of selenite with hepatocytes, protein-bound Se was the predominant form (80-90% of the cellular Se) present. Subcellular fractionation indicated that most of the radioactivity was present in the cytosol when hepatocytes were incubated with 75Se-selenite. The uptake of 75Se by isolated rat hepatocytes was linear with selenite concentration up to the highest amount tested, 200 microM. Sulfite inhibited the uptake of selenite by hepatocytes.

Absorption↗

Rat plasma selenoprotein P properties and purification.

A selenoprotein in rat plasma, selenoprotein P, was fractionated and characterized. Plasma collected from rats 3 h post injection of 75SeO3(2-) contained one 75Se-labeled protein, selenoprotein P. Selenoprotein P was fractionated using salt precipitation, Affi-Gel Blue, and DEAE chromatography. The 75Se-containing subunit of selenoprotein P was purified to 90% homogeneity using SDS-polyacrylamide gel electrophoresis followed by electroelution. This isolation resulted in an 850-fold purification of the 75Se-containing subunit of selenoprotein P with a 15% yield of 75Se radioactivity. The molecular weight of selenoprotein P in plasma was 98,000. The 75Se-containing subunit of selenoprotein P had a molecular mass of 57 kDa as determined by SDS-polyacrylamide gel electrophoresis. Isoelectric focusing under nondenaturing conditions resulted in a band of 75Se radioactivity at pH 5.4. A comparison of Coomassie Blue- and silver-staining properties of selenoprotein P in SDS-polyacrylamide gels was made. Reverse-phase HPLC and Sephadex G-50 chromatography of tryptic peptides of the 57 kDa subunit of selenoprotein P yielded several peaks of 75Se radioactivity. These results indicate that 75Se is present in several locations within the 57 kDa subunit of selenoprotein P.

Animals↗

Relationship between plasma adrenocorticotrophic hormone and cortisol concentrations in chronic alcoholic patients with depression.

In 20 hospitalized male chronic alcoholic patients, plasma adrenocorticotrophic hormone (ACTH) levels were estimated by radioimmunoassay and cortisol levels by the competitive protein binding (CPB) method with radioactive selenium-75 on admission and during abstinence along with rating of the degree of depression immediately after the acute phase of the ethanol withdrawal syndrome was over. Duration of drinking ranged from 5 to 25 years and average daily ethanol intake was between 100 and 150 g. Plasma ACTH levels were found to be raised in drinking chronic alcoholics. There was a positive and statistically significant correlation between depression ratings and plasma ACTH concentrations (r = + 0.379; P less than 0.05). In chronic alcoholism the negative feedback mechanism seems to be disturbed between plasma ACTH and cortisol levels which are not normalised after 1 week of total abstinence. Chronic ethanol ingestion might have a direct stimulatory effect on the adrenal cortex leading to dysregulation of the hypothalamo-pituitary-adrenocortical axis.

Adrenocorticotropic Hormone↗

Fast chemical synthesis of [75Se]L-selenomethionine.

A fast chemical synthesis of high specific activity [75Se]L-selenomethionine (10 Ci/mmol--370 GBq/mmol) is described with a view to 73Se labeling and PET studies. The overall radiochemical yield of the preparation is better than 80%. The purification method uses commercially available reverse phase HPLC columns and 9% NaCl as mobile phase. The final labeled compound is obtained in less than 3 h and the chemical, radiochemical and optical purities of the L-isomer are higher than 99.0%.

Humans↗

Chemical processing for production of no-carrier-added selenium-73 from germanium and arsenic targets and synthesis of L-2-amino-4-([73Se]methylseleno) butyric acid (L-[73Se]selenomethionine).

The Ge(4He, xn) and 75As(p, 3n) reactions were compared as the best potential routes for routine production of selenium-73 (73Se) for medical applications. With 26 MeV alpha particles, available with compact cyclotrons, the first reaction required an enriched 70Ge target of sodium metagermanate to give a production yield of 1 mCi/microAh (0.037 GBq/microAh) in a 105 mg/cm2 target. With 55 MeV protons the As(p, 3n) reaction on natural arsenic yielded 20 mCi/microAh (0.74 GBq/microAh) in a 685 mg/cm2 target. A simple method was developed and optimized for both targets in order to isolate and purify the no-carrier-added selenium in the elemental form with a radiochemical yield greater than 75% in less than 90 min. An automated radiochemical processing unit has been designed for the routine production of 100-150 mCi (3.7-5.5 GBq) batches of carrier-free 73Se ready for radiopharmaceutical labeling. 30 mCi (1.11 GBq) (EOS) of L-2-amino-4-([73Se]methylseleno) butyric acid (L-[73Se]selenomethionine) ready for injection with a specific activity of 5 Ci/mmol (185 GBq/mmol) (EOS) were obtained through a fast chemical synthesis. Radiation absorbed dose estimates for L-[73Se]selenomethionine have been determined. A value of 0.70 rem/mCi (0.19 mSv/MBq) administered was calculated for the risk from irradiation in man. The first human PET investigation with [73Se]selenomethionine showed a very good delineation between liver and pancreas.

Arsenic↗

Biodistribution of bis-[beta-(N,N,N-trimethylamino)ethyl]selenide-75Se diiodide, a potential articular cartilage imaging agent.

In an effort to develop a specific radiodiagnostic agent for articular cartilage imaging, we have investigated the biodistribution of bis[beta-(N,N,N-trimethylamino)ethyl]-selenide-75Se diiodide (75Se BISTAES) in rabbits. At an intravenous dose of 5 mg/kg, the greatest localization of the compound occurred in articular cartilage 15 min after injection. The compound was excreted rapidly in the urine. The results suggest that 75Se BISTAES has potential clinical use as an articular cartilage imaging agent.

Animals↗

The biodistribution of [75Se]bis-[beta-(N,N,N-trimethylamino)ethyl]selenide diiodide in adult guinea pigs.

The biodistribution of [75Se]BISTAES was studied in guinea pigs. A higher concentration of radioactivity was observed in articular cartilage than in other tissues or organs. A minimal amount of radioactivity was found in the blood, muscle and bone. The compound was excreted rapidly in urine. The target to background ratios were encouraging. [75Se]BISTAES has potential as an articular cartilage imaging agent and further studies in osteoarthritic animals are merited.

Animals↗

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 gamma-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 filled 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.

Body Burden↗

Synthesis and tissue distribution of four Se-labeled tertiary amines, potential brain pH imaging agents.

Four new tertiary amines: bis(3-N,N-dimethyl aminopropyl)selenide (PROMOSE), bis(3-N-(morpholino)propyl)selenide, N-methyl-selenomorpholine and N-phenyl-selenomorpholine structurally related to MOSE proposed by Kung and Blau, have been labeled through a radiochemical procedure suitable for both 75Se and 73Se. The radiochemical yields of the carrier added synthesis ranged between 64 and 85% for the four 75Se labelings and was 64% EOB time corrected for [73Se]PROMOSE. The chemical and radiochemical purities were higher than 99% after chromatographic purifications. The n-octanol/phosphate buffer partition coefficients (P) were measured at various pH (6.5-8) for each compound and the tissue distributions of PROMOSE in rats were also carried out. The experimental results showed a good correlation between the P = f(pH) function and the in vivo behaviour of the considered compound. PROMOSE was selected for further investigations as a brain pH indicator.

Amines↗