Stimulation of mucosal uptake of selenium from selenite byl-cysteine in sheep small intestine

The influence of cysteine (Cys) on mucosal uptake of 75Se-labeled selenite in sheep midjejunum was investigated using a short-term uptake technique. L-Cys (concn.: 1.0 mmol/L) significantly stimulated uptake of Se from selenite (concn.: 10 mumols/L). The stimulatory effect of L-Cys on mucosal uptake of Se from selenite was Na(+)- and pH-dependent. In the absence of Na+, or at an acidic pH (5.0), the stimulatory effect of L-Cys was abolished. L-alanine and L-lysine, but not L-glutamic acid inhibited uptake of Se from selenite in the presence of L-Cys. Preincubation of mucosal preparations with 10 mmol/L L-Cys produced enhanced mucosal uptake of Se from selenite. It is concluded from these results that L-Cys stimulates absorption of Se from selenite probably by generation of selenodicysteine and maybe cysteine selenopersulfide that are subsequently transported across the intestinal brush border membrane by Na(+)-dependent amino acid carriers. Furthermore, intracellular generation of selenodicysteine might contribute to the uptake of Se from selenite by maintaining the concentration gradient for diffusive uptake of selenite.     

Speciation and metabolism of selenium injected with 82Se-enriched selenite and selenate in rats

Selenate and selenite injected intravenously into rats were speciated by the HPLC–ICP MS method with use of an enriched stable isotope as the tracer. In dose–relation experiments, ⁸²Se-enriched selenate or selenite was injected intravenously into male Wistar rats of 8 weeks of age (three rats/group) at single doses of 10, 25, 50, 100 and 200 μg/kg body weight for the selenate group, and 2, 5, 10, 25 and 50 μg/kg body weight for the selenite group. The animals were sacrificed 1 or 24 h later, and the concentrations and distributions of ⁸²Se in the liver, kidneys, serum, and urine remaining in the bladder or 24-h urine were determined. In time-course experiments, ⁸²Se-enriched selenate and selenite were injected at doses of 50 and 10 μg/kg body weight, respectively, and the animals were sacrificed 5, 15, 30, 60 and 180 min later. It was suggested that selenate is directly taken up by the liver with an efficiency of approximately 1/2 compared with selenite, the latter being taken up by the liver after being metabolized to selenide in red blood cells. Although selenate and selenite were metabolized differently in the bloodstream, and also a part of only selenate was excreted directly into the urine, the ⁸²Se taken up by the liver was shown to be metabolized in a manner indistinguishable between selenate and selenite. ⁸²Se of selenite origin but not of selenate origin was suggested to undergo redox reaction in the bloodstream. These results suggest that although parenteral selenate is utilized less efficiently by the body, it is utilized in the liver in a similar manner to selenite much more safely.     

Detection of prion protein particles in blood plasma of scrapie infected sheep

Prion diseases are transmissible neurodegenerative diseases affecting humans and animals. The agent of the disease is the prion consisting mainly, if not solely, of a misfolded and aggregated isoform of the host-encoded prion protein (PrP). Transmission of prions can occur naturally but also accidentally, e.g. by blood transfusion, which has raised serious concerns about blood product safety and emphasized the need for a reliable diagnostic test. In this report we present a method based on surface-FIDA (fluorescence intensity distribution analysis), that exploits the high state of molecular aggregation of PrP as an unequivocal diagnostic marker of the disease, and show that it can detect infection in blood. To prepare PrP aggregates from blood plasma we introduced a detergent and lipase treatment to separate PrP from blood lipophilic components. Prion protein aggregates were subsequently precipitated by phosphotungstic acid, immobilized on a glass surface by covalently bound capture antibodies, and finally labeled with fluorescent antibody probes. Individual PrP aggregates were visualized by laser scanning microscopy where signal intensity was proportional to aggregate size. After signal processing to remove the background from low fluorescence particles, fluorescence intensities of all remaining PrP particles were summed. We detected PrP aggregates in plasma samples from six out of ten scrapie-positive sheep with no false positives from uninfected sheep. Applying simultaneous intensity and size discrimination, ten out of ten samples from scrapie sheep could be differentiated from uninfected sheep. The implications for ante mortem diagnosis of prion diseases are discussed.     

In vitro studies on methyl mercury distribution in human blood

Studies comparing the methyl mercury (mHg) content of maternal and newborn blood have shown increased levels in the newborn. This has been attributed to facilitated transplacental diffusion because of high fetal hematocrit (Hct). This study shows the converse, that the diffusion of mHg diminishes progressively with increasing Hct. The diffusion of m203Hg across a Millipore membrane (0.45 microns) separating compartments A and B of a diffusion cell was studied. When both compartments contained saline or plasma alone, equilibration from A to B occurred in 5 h. Introduction of human red blood cells (RBC) in saline (Hct 20%) into B resulted in a twofold increase in diffusion of mHg when compared to saline alone. Increasing Hct in saline in compartment B resulted in a progressive decrease in diffusion (r = -0.95, P less than 0.001). The presence of RBC in plasma (Hct 20%) in B resulted in a 70% decrease in diffusion; with increasing Hct, diffusion was further reduced (r = -0.95, P less than 0.001). Direct addition of mHg to RBC in saline resulted in 98% RBC uptake. Increasing concentrations of plasma (at a constant Hct) resulted in a progressive decrease in RBC uptake. In undiluted plasma at Hct 14%, RBC uptake of mHg was 35%. Plasma electrophoresis showed that much of the mHg was associated with a high-molecular-weight lipoprotein fraction. Plasma components appear to be important in the distribution of mHg in blood, and may be a factor in the relatively higher blood levels in the fetus.     

Progesterone and 20 alpha-dihydroprogesterone in sheep: a model of their distribution and metabolism.

The rates of metabolism of progesterone and 20alpha-dihydroprogesterone (20alpha-diHP) in sheep have been measured during and after the infusion of tracer amounts of [3H]progesterone. There were significant differences in the blood concentration of [3H]progesterone between experiments, but these were not attributable to the stage of gestation or to the difference between pregnant and non-pregnant animals. The mean (+/- S.E.M.) metabolic clearance rate of progesterone was 3-277 +/- 0-239 litres blood/min. The simplest model of the distribution of progesterone was one containing two pools, V1[P] and V2[P], where [p] is the blood concentration of progesterone, and in 23 experiments on 7 sheep the mean pool dimensions were 7-8[P] mug and 70[P] mug, respectively. This model was developed to include the formation of 20alpha-diHP from progesterone. Progesterone appeared to be the major source of 20alpha-diHP, though this did not seem to be an obligatory metabolite. The parameters obtained gave comparably low residual deviations for both labelled steroids and were consistent with other observations made on progesterone clearance.     

Interactions of cadmium and selenium in rat plasma in vivo and in vitro.

75Se and 109Cd tracers were used to study the binding of Se and Cd to plasma proteins at various SeO32- doses and times upt to 24 h after the simultaneous subcutaneous administration of SeO32- markedly increased both Se and Cd plasma levels over that in control animals. Gel permeation chromatography of plasma indicated that at all times up to 24 h Cd and Se were bound in an atomic ratio of approx. 1 : 1 in 330 000 and 130 000 dalton fractions. From 4 to 24 h, Cd and Se appeared in the 420 000 dalton fraction, also with an atomic ratio of approx. 1 : 1. The 330 000 dalton molecules appeared to have a maximal binding capacity for the Cd-Se complex at a concentration of approx. 30 mumol/ml of plasma, while the 130 000 and 420 000 dalton molecules show a higher binding capacity. Studies in vitro revealed that SeO32- does not interact directly with Cd and plasma proteins. It is metabolized by erythrocytes to a form that interacts in an atomic ratio of 1 : 1 with Cd to form a protein-bound complex of 130 000 daltons.     

In vitro thymidine labelling in human and porcine growth plates

An in vitro technique has been used to label dividing cells in the growth plates of human bones with tritiated thymidine. The patterns of labelling in autoradiographs of human plates are described and values given for the labelling index, the number of cells in the proliferation zones and the heights of hypertrophied cells. In two of the four subjects no labelled cells were found in the growth plates and possible causes for these failures are discussed. In vitro labelling data on five porcine growth plates are also presented for comparison with the human data. In both structure and cell kinetics the epiphyseal cartilage plate in the pig is intermediate between the human and rodent plates.     

In vitro interaction of fenretinide with plasma retinol-binding protein and its functional consequences.

The synthetic retinoid fenretinide (4-HPR; N-[4-hydroxyphenyl] all-trans-retinamide) interacts with plasma apo-retinol-binding protein (RBP) to form a tight complex (K'd approximately 0.2 microM) which does not exhibit binding affinity to transthyretin (TTR). Therefore, a substantial modification of the retinol hydroxyl group does not appear to affect the interaction with RBP but does drastically interfere with the protein-protein recognition. The remarkable early reduction in plasma retinol level induced by fenretinide administration may be associated with the high binding affinity of this retinoid to RBP and to its interference with the RBP-TTR complex formation.     

In vitro synthesis of glutathione peroxidase from selenite. Translational incorporation of selenocysteine

The synthesis of glutathione peroxidase from [75Se]selenite was studied in slices and cell-free extracts from rat liver. The incorporation of [75Se]selenocysteine at the active site was detected by carboxymethylation and hydrolysis of partially purified glutathione peroxidase (glutathione:hydrogen peroxide oxidoreductase, EC 1.11.1.9) in the presence of [3H]selenocysteine and subsequent amino acid analysis. The synthesis of glutathione peroxidase in slices was inhibited by cycloheximide or puromycin and 75Se was incorporated from [75Se]selenite into free selenocysteine and selenocysteyl tRNA. Increasing concentrations of selenocystine caused a progressive dilution of the 75Se and a corresponding decrease in glutathione peroxidase labeling. In cell-free systems, [75Se]selenocysteyl tRNA was the best substrate for glutathione peroxidase synthesis. These results indicate the existence in rat liver of the de novo synthesis of free selenocysteine and a translational pathway of selenocysteine incorporation into glutathione peroxidase.     

Effects of suramin on the immune responses to sheep red blood cells in mice. II. In vitro studies

The effect of Suramin on the secondary in vitro response to sheep erythrocytes (SRBC) was studied. Spleen cells from mice which were treated with Suramin immediately prior to sensitization with SRBC failed to respond to an in vitro SRBC challenge. This Suramin-induced immunosuppression is not related to a defect in macrophage or B-cell function(s). Suramin does not interfere with the induction by SRBC of radioresistant and radiosensitive helper-T-cell subpopulations. Cell separation studies, using wheat germ agglutinin, showed radiosensitive helper-T-cell function in the nonagglutinated fraction while the radioresistant helper activities are carried out by the agglutinated subpopulation. Evidence is presented that Suramin administration results in a suppressive T-cell activity which can be demonstrated in the subpopulation agglutinated by wheat germ agglutinin. The role of such suppressive T cells in the inhibitory effect exerted by Suramin on the cell-mediated delayed-type hypersensitivity response to SRBC is discussed.     

In vitro lipid metabolism in the rat pancreas. I. Basal lipid metabolism.

1. The in vitro basal lipid metabolism of rat pancreatic fragments was compared with that in adipose tissue fragments and liver slices. 2. [1-14C]Acetate added to the media was mostly incorporated into palmitic acid and to a lesser extent into oleic acid. In addition, pancreatic tissue exhibited a marked capacity for elongation of polyunsaturated fatty acids by [1-14C]acetate and resulting desaturation when compared to adipose tissue and liver. 3. Data obtained in the presence of [U-14C]glucose, [1-14C]palmitate and 3H20 indicate that acetyl-CoA derived from glucose and from beta-oxidation of fatty acids contributed to de novo lipogenesis. 4. Oxidation of [1-14C]palmitic acid was 9-13 times higher in the pancreas than in adipose tissue or liver when expressed on a wet weight basis. 5. The fatty acid moiety of pancreatic glycerolipids could be derived from de novo synthesis, fatty acids added to the medium, or from fatty acids formed from the hydrolysis of endogenous lipids. The glycerol moiety could be derived either from glucose, or directly from glycerol through participation of glycerol kinase.