Plasma selenium in specific and non-specific forms

Selenium is present in plasma and tissues in specific and non-specific forms. The experiments reported here were carried out to clarify some factors that affect these forms of the element in plasma. A selenium-replete human subject was given 400 microg of selenium daily for 28 days as selenomethionine and, in a separate experiment, as selenate. The selenomethionine raised plasma and albumin selenium concentrations. Selenate did neither. The molar ratio of methionine to selenium in albumin was approximately 8000 under basal and selenate-supplemented conditions but 2800 after selenomethionine supplementation. This demonstrates that selenium from selenomethionine, but not selenium from selenate, can be incorporated into albumin, presumably as selenomethionine in the methionine pool. Selenocysteine incorporation into albumin was studied in rats using (75)Se-selenocysteine. No evidence was obtained for incorporation of (75)Se into albumin after exogenous administration or endogenous synthesis of (75)Se-selenocysteine. Thus, selenocysteine does not appear to be incorporated non-specifically into proteins as is selenomethionine. These findings are in support of selenomethionine being a non-specific form of selenium that is metabolized as a constituent of the methionine pool and is unaffected by specific selenium metabolic processes. No evidence was found for non-specific incorporation of selenium into plasma proteins when it was administered as selenate or as selenocysteine. These forms of the element appear to be metabolized by specific selenium metabolic processes.     

Induction of retrovirus gene expression by selenium compounds

Sodium selenite, sodium selenate, selenium oxide, selenophypoxanthine, selenopurine, selenocysteine, selenoethionine and selenomethionine were tested for their ability to induce endogenous retrovirus expression in cultured AKR mouse embryo fibroblasts. All except selenoethionine were highly toxic to the cells. Only selenomethionine however, had the ability to induce virus expression under the conditions used. The level of virus induction (plaque-forming-units/10(5) cells) was roughly proportional to dose over the range of concentrations from 0.25 mM to 5.0 mM. Induction was best observed when a treatment duration of 48 h was used and required the treatment of actively dividing cells. The induction and the cytotoxic effects of selenomethionine could be abrogated by simultaneous treatment with methionine. A ratio of methionine to selenomethionine of 1:10 inhibited induction by approx. 60% while equivalent amounts of methionine inhibited selenomethionine-mediated induction by greater than 96%, indicating that methionine was more efficiently recognized by the cells than was selenomethionine. A possible mechanism for selenomethionine induction involving the production of undermethylated DNA is presented.     

Interactions of Methionine and Selenomethionine with Methionine Adenosyltransferase and Ethylene-generating Systems

Since selenomethionine appears to be a better precursor of ethylene in senescing flower tissue of Ipomoea tricolor and in indole acetic acid-treated pea stem sections than is methionine (Konze JR, N Schilling, H Kende 1978 Plant Physiol 62: 397-401), we compared the effectiveness of selenomethionine and methionine to participate in reactions which may be connected to ethylene biosynthesis. Evidence is presented that selenomethionine is also a better substrate of methionine adenosyltransferase (ATP: methionine S-adenosyltransferase, EC 2.5.1.6) from I. tricolor, the V_max for selenomethionine being twice as high as that for methionine. The affinity of the enzyme is higher for methionine than for selenomethionine, however. Methionine added to flower tissue together with selenomethionine inhibits the enhancement of ethylene synthesis by the seleno analog. Likewise, methionine reduces the high, selenomethionine-dependent reaction rates of methionine adenosyltransferase from I. tricolor flower tissue. On the other hand, selenomethionine is less effective as an ethylene precursor than is methionine in model systems involving oxidation by free radicals. It was concluded that activation of methionine by methionine adenosyltransferase and formation of S-adenosylmethionine are more likely to be involved in ethylene biosynthesis than is oxidation of methionine by free radicals.     

Characteristic chain-end racemization behavior during photolysis of poly(L-lactic acid)

Photolysis of poly(L-lactic acid) (PLLA) has many unclear points, such as the degradation mechanism, kinetics, products, and racemization mechanism. To clarify these features of PLLA photolysis, we examined the relationship between photolysis and racemization. The hexad stereosequential analysis of photodegraded PLLA was investigated to specify the racemized positions within a chain in comparison with hydrolysis and thermal degradation. Results from (13)C NMR spectra of UV-irradiated PLLA samples indicated that the samples have racemized d-lactate units at chain ends. From the comparison of racemization behavior among photolysis, hydrolysis, and thermal degradation, it was confirmed that the preferential racemization behavior of each of these three degradation processes is characteristic and distinct, being identified as chain-end racemization, poor racemization, or internal-unit racemization, respectively. The characteristic chain-end racemization behavior of photolysis was first confirmed in this study.     

Simultaneous use of 1-hydroxybenzotriazole and copper(II) chloride as additives for racemization-free and efficient peptide synthesis by the carbodiimide method.

In the carbodiimide mediated coupling of Z-Gly-L-Val-OH with H-L-Val-OMe in DMF, the simultaneous use of HOBt and copper(II) chloride as additives was found to give the desired peptide in a high yield without racemization. In the presence of HOBt, reducing the amount of copper(II) chloride produced a higher yield. Besides improving the coupling efficiency as compared with the case using copper(II) chloride alone as an additive, the present procedure offered another advantage for racemization suppression. Thus, even for the couplings where a low level of racemization was observed in the presence of copper(II) chloride, the simultaneous addition of HOBt and copper(II) chloride resulted in the elimination of racemization. The effectiveness of this new procedure using the two carbodiimide additives in the synthesis of biologically active peptides was assessed by the preparation of a protected Leu-enkephalin. In the 4 + 1 segment condensation using HOBt and copper(II) chloride simultaneously as additives, no racemization was detected and the yield was high enough. The elimination of racemization and improvement of coupling efficiency produced by the present procedure can be attributable to a reduced tendency for the activated forms of the carboxyl component to form a 5(4H)-oxazolone by the action of HOBt, and to the prevention of racemization by copper(II) chloride of the small amount of the oxazolone formed which is not eliminated by the action of HOBt alone.     

Selenomethionyl analog of recombinant human choriogonadotropin

Selenomethionyl and high mannose type analog of recombinant human choriogonadotropin (hCG) to solve the crystallization and phase problems has been obtained by gene transfer methodology. SF9 insect cells were infected with the recombinant viruses containing hCG alpha and hCG beta cDNAs in selenomethionine containing methionine-free Grace's medium. The selenomethionyl hCG (SehCG) was purified from the culture medium by one step immunoaffinity chromatography using an immobilized monoclonal antibody against hCG beta. The presence of selenomethionine was demonstrated by amino acid analysis of SehCG. The amino acid composition indicated that more than 84% of methionine residues were substituted by selenomethionine. Its sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis yielded a single 38-kDa protein band under nonreducing conditions. The carbohydrate analysis of SehCG was consistent with the presence of four N-linked high mannose type carbohydrates and four O-linked simple disaccharide chains. The in vitro immunological and biological studies of SehCG indicated that selenomethionine substitution had no effect on the immunopotency, receptor binding, and steroidogenic activities of the hormone.     

Kinetics of racemization of (+)- and (−)-diethylpropion: Studies in aqueous solution,with and without the addition of cyclodextrins,in organic solvents and in human plasma

The configurational stability of (+)- and (−)-diethylpropion [(+)- and (−)-2-(diethyl)-1-phenyl-1-propanone or (+)- and (−)-DEP ] was investigated systematically from chemical, pharmaceutical, and pharmacological aspects. The enantiomeric ratio was monitored directly with a recently developed stability-indicating enantioselective HPLC method. In aqueous solutions, the rate of racemization increased non-linearly with increasing pH and with increasing phosphate buffer concentration. The racemization rate showed a positive slope with increasing temperature and decreasing ionic strength. The racemization rates of (+)- and (−)-DEP in the presence of cyclodextrins (CDs) did not differ significantly. CDs that were added to (+)- and (−)-DEP in a molar ratio 5:1 showed the following effects after dissolution in 10 mM phosphate buffer (final pH 6.7): sulfobutyl ether-β-CD (SBE-β-CD) and methylated-β-CD (Me-β-CD) retarded racemization; whereas hydroxypropyl-β-CD (HP-β-CD), acetyl-γ-CD (Ac-γ-CD), acetyl-β-CD (Ac-β-CD), γ-CD, and β-CD showed a weak destabilising effect. In contrast to the described CDs, α-CD distinctly accelerated the rate of racemization. The configurational stability of (+)- and (−)-DEP was also studied under physiological conditions. The half-life of racemization in heparinised human plasma was for both enantiomers determined to be approximately 23–25 min. In phosphate buffer (10 mM, pH 7.4), rac-DEP showed a high, but unselective affinity towards human α₁-acid glycoprotein (orosomucoid) immobilised on silica (Chiral AGP). The rate of racemization of the free base of (−)-DEP dissolved in organic solutions generally increases with the polarity of the solvating agent. Chirality 10:307–315, 1998. © 1998 Wiley-Liss, Inc.     

Methionine Changes in Bacteriorhodopsin Detected by FTIR and Cell-Free Selenomethionine Substitution

Bacteriorhodopsin (BR) is an integral membrane protein, which functions as a light-driven proton pump in Halobacterium salinarum. We report evidence that one or more methionine residues undergo a structural change during the BR→M portion of the BR photocycle. Selenomethionine was incorporated into BR using a cell-free protein translation system containing an amino acid mixture with selenomethionine substituted for methionine. BR→M FTIR difference spectra recorded for unlabeled and selenomethionine-labeled cell-free expressed BR closely resemble the spectra of in vivo expressed BR. However, reproducible changes occur in two regions near 1284 and 900 cm⁻¹ due to selenomethionine incorporation. Isotope labeled tyrosine was also co-incorporated with selenomethionine in order to confirm these assignments. Based on recent x-ray crystallographic studies, likely methionines which give rise to the FTIR difference bands are Met-118 and Met-145, which are located inside the retinal binding pocket and in a position to constrain the motion of retinal during photoisomerization. The assignment of methionine bands in the FTIR difference spectrum of BR provides a means to study methionine-chromophore interaction under physiological conditions. More generally, combining cell-free incorporations of selenomethionine into proteins with FTIR difference spectroscopy provides a useful method for investigating the role of methionines in protein structure and function.     

Selenium supplementation of infant formula: uptake and retention of various forms of selenium in suckling rats

Formula-fed infants often have lower serum selenium levels than breast-fed infants. Although no deleterious effects have been correlated to this finding, supplementation of formula with selenium is considered. In this study, we investigated the uptake and retention by suckling rat pups of ⁷⁵Se from selenite, selenate, and selenomethionine added to infant formula. The molecular distribution of ⁷⁵Se in liver, kidney, intestine, and plasma was followed by gel-filtration chromatography on Superose 12. ⁷⁵Se-uptake was most rapid from selenomethionine (70% at 1 hr), followed by selenate (51%) and selenite (29%). This difference was explained by a higher retention of ⁷⁵Se in the stomach and small intestinal wall of pups given selenite supplement. Plasma distribution of ⁷⁵Se as studied by gel filtration was also different, with a higher proportion of ⁷⁵Se from selenomethionine being protein-bound than from selenite or selenate. Similarly, a larger proportion of ⁷⁵Se from selenomethionine became protein-bound in the liver than from selenite or selenate. In conclusion, although whole body retention after 24–48 hr was similar, the metabolic fate of selenium varies considerably with the form of selenium added to formula. Further studies are needed to study the long-term consequences of selenium accumulated in different body compartments.     

Route-dependent pharmacokinetics, distribution, and placental permeability of organic and inorganic selenium in hamsters

Inorganic selenium (Se) salts (selenite and selenate oxyanions) and the organic selenoamino acids (selenomethionine and seleniferous grains) are teratogenic and embryolethal in domestic and wild birds. Selenium bioaccumulation has been held responsible for reproductive failure among waterfowl at the Kesterson Reservoir (California), the Ouray and Stewart Lake Wildlife Refuges (Utah), and the Carson Sink (Nevada). Anecdotal field and controlled laboratory reports have implicated Se exposure in mammalian embryotoxicity (including human), but developmental toxicity studies in hamsters failed to demonstrate an adverse response, except at maternally toxic doses (Ferm et al., Reprod. Toxicol., in press). Uptake, distribution, and elimination of Se after a single bolus equimolar dose (60 mumol/kg) of selenate or selenomethionine by oral or intravenous administration were compared using day 8 pregnant hamsters. Intravenous selenate was eliminated ten times more rapidly from maternal plasma than oral selenate, but concentrated in liver, kidney, and placenta to the same degree. Intravenous (iv) L-selenomethionine achieved lower maximum circulating total [Se], but it was eliminated more slowly than iv selenate. Larger areas under the plasma and peripheral tissue [Se]:time curve (AUC) after oral or parenteral selenomethionine than after equimolar selenate were consistent with previous studies in rodents and in humans. Embryonic [Se] plateaued at 3 nmol/g after selenate, but embryonic [Se] after selenomethionine continued to accumulate (80 nmol/g) as gestation progressed. The lack of a teratogenic response in hamsters at doses of either selenate or selenomethionine less than those associated with maternal intoxication cannot be attributed to lack of Se accumulation in early embryonic and placental tissue.     

Effect of copper(II) chloride on suppression of racemization in peptide synthesis by the mixed anhydride and related methods.

In segment couplings by the mixed anhydride method using isobutyloxycarbonyl chloride, the use of copper(II) chloride as an additive suppressed racemization completely in the same manner as in the carbodiimide method reported previously. This was confirmed by employing a number of couplings between Z-dipeptides and amino acid esters. The racemization-suppressing effect of other compounds were also evaluated by employing one of these model couplings to be at best only limitedly effective. Copper(II) chloride was effective also in the related method using EEDQ. Thus, in the couplings where a low level of racemization was observed without an additive, the addition of copper(II) chloride eliminated racemization even at ambient temperature where EEDQ is usually used. The effectiveness of copper(II) chloride was confirmed also in the BOP-Cl method. In the presence of HOBt racemization was reduced to a low but still detectable level, while it was suppressed completely by the addition of copper(II) chloride.     

Histidine racemization in the synthesis of an analog of the luteinizing hormone releasing factor. Investigation by HPLC and enzymatic methods

To investigate histidine racemization in the synthesis of a LHRH analog, (D-Trp)6-LHRH2-10 was built up by stepwise elongation of the sequence 3-10 using the solid phase technique on a 1% cross-linked chloromethyl polystyrene. For the whole synthesis the tert.-butyloxycarbonyl (BOC) group was used for temporary N-terminal protection. To protect the pi-nitrogen in histidine the benzyl-oxymethyl (BOM) group was utilized. The condensation position in the (D-Trp)6-LHRH analog was chosen so as to be able to investigate the racemization of histidine. We coupled BOC-His(BOM) with the (D-Trp)6-LHRH3-10 fragment using three different activating agents, mixed anhydride, carbonyldiimidazole (1 equiv.)/1-hydroxybenzotriazole (2 equiv.) and dicyclohexylcarbodiimide/1-hydroxybenzotriazole. The racemization was investigated by enzymatic digestion and by HPLC. For HPLC, (D-His(BOM)2-(D-Trp)6-LHRH2-10 was also synthesized. It could be proved that practically no racemization occurs during the actual peptide synthesis. The small amount (1%) of D-histidine found is due to racemization in the synthesis of BOC-His(BOM).     

Failure of selenomethionine residues in albumin and immunoglobulin G to protect against peroxynitrite.

Selenomethionine has been suggested to protect against peroxynitrite by quenching it in vivo. Selenomethionine is distributed randomly in the methionine pool. Albumin and IgG were purified from plasma of a human being before and after 28 days of supplementation with 400 microg selenium/day as selenomethionine. The albumin contained 1 selenium atom, presumably as selenomethionine, per 8000 methionine residues before supplementation and 1 per 2800 after supplementation. Although this ratio suggested that selenomethionine would not have as great an effect in quenching peroxynitrite as would methionine, direct testing of the albumin and IgG fractions was carried out to assess the ability of these proteins to prevent peroxynitrite oxidation of dihydrorhodamine 123 to rhodamine 123. The ability of the albumin preparations to resist nitration of tyrosine residues was also assessed. The high-selenomethionine preparations of the proteins had no greater effect in quenching the peroxynitrite than did the normal-selenomethionine preparations. These results do not support the proposal that selenomethionine in proteins contributes to in vivo protection against peroxynitrite.     

Resolution and stability of oxazepam enantiomers

A solvent mixture containing dioxane, acetonitrile, and hexane was found to be suitable as a mobile phase to resolve oxazepam enantiomers by chiral stationary phase high performance liquid chromatography using covalent Pirkle columns. The resolved oxazepam enantiomers in this solvent mixture had a racemization half-life greater than 3 days at 23°C. When desiccated at 0°C as dried residue, OX enantiomers were stable for at least 50 days with less than 2% racemization. The conditions which stabilized OX enantiomers significantly facilitated the determination of racemization half-lives of OX enantiomers in a variety of aqueous and nonaqueous solvents and at different temperatures. © 1992 Wiley-Liss, Inc.     

Differential racemization of aspartate and serine in human myelin basic protein

L-Aspartate and L-serine were found to undergo amino acid racemization in brain myelin basic protein (MBP) of aging humans. The observed racemization was different in each chromatographically purified MBP isoform. Pepsin digestion of MBP produced three peptides, each of which exhibited different degrees of racemization of the same amino acids. MBP isolated by the same method from rat and guinea pig brain showed little accumulation of D-amino acids. Total MBP isolated from SDS-polyacrylamide disc gel electrophoresis of total human myelin proteins (delipidated myelin) was racemized to the same extent as purified MBP, indicating that the racemization observed was not an artifact of the isolation procedure. Myelin proteolipid protein from the same gel was racemized approximately half as much as MBP. The age and environment of the aspartates and serines in myelin proteins may strongly affect their observed racemizations.     

In vivo incorporation of selenomethionine in proteins using Pseudomonas aeruginosa as expression host: case study-the outer membrane receptor FpvA

The number of protein structures solved using multiwavelength anomalous diffraction methods coupled with selenomethionine substitution has grown dramatically over the last years. We show using the outer membrane pyoverdin receptor FpvA that Pseudomonas aeruginosa can be used for producing proteins with a high level of selenomethionine incorporation. To circumvent problems encountered with mass spectroscopy analysis of purified membrane proteins, in-gel trypsin digestion of FpvA coupled with MALDI mass spectrometry analysis of the resulting peptides was used to determine the extent of selenomethionine incorporation. Selenomethionine incorporation greater than 95% was achieved using P. aeruginosa as an overexpression system.     

Selenium Alleviates Porcine Nephrotoxicity of Ochratoxin A by Improving Selenoenzyme Expression In Vitro

Ochratoxin A (OTA), a mycotoxin, is a potent nephrotoxin in humans and animals. Selenium (Se) is an essential micronutrient for humans and animals, and plays a key role in antioxidant defense. To date, little is known about the effect of Se on OTA-induced nephrotoxicity. In this study, the protective effects of selenomethionine against OTA-induced nephrotoxicity were investigated using the porcine kidney 15 (PK15) cells as a model. The results showed that OTA induced nephrotoxicity in a dose-dependent manner. Se at 0.5, 1, 2 and 4 μM had significant protective effects against OTA-induced nephrotoxicity. Furthermore, selenomethionine enhanced the activity and mRNA and protein expression of glutathione peroxidase 1 (GPx1), mRNA expression of GPx4, and mRNA expression of thioredoxin reductase 1 in the presence and absence of OTA. Among them, promoting effect of selenomethionine on GPx1 was maximal. Knock-down of GPx1 by using a GPx1-specific siRNA eliminated the protective effects of selenomethionine against OTA-induced nephrotoxicity. The results suggest that selenomethionine alleviates OTA-induced nephrotoxicity by improving selenoenzyme expression in PK15 cells. Therefore, selenomethionine supplementation may be an attractive strategy for protecting humans and animals from the risk of kidney damage induced by OTA.     

Unexpected racemization of proline or hydroxy-proline phenacyl ester during coupling reactions with Boc-amino acids.

When L-proline or O-benzyl-trans-4-hydroxy-L-proline phenacyl ester was coupled with Boc-amino acids in dimethylformamide using water-soluble carbodiimide (WSCI) in the presence of anhydrous 1-hydroxybenzotriazole (HOBt) as coupling reagents, extensive racemization was observed at the C alpha of the proline or hydroxy-proline residue. The extent of racemization was measured by HPLC after the coupling with Boc-L-Leu-OH in the presence or absence of HOBt. The extent of racemization increased when HOBt was added to the reaction mixture, but greatly decreased when it was not, indicating that HOBt was needed for inducing racemization. Almost no racemization was observed when the coupling reaction was carried out by the mixed anhydride procedure in tetrahydrofuran or by the carbodiimide method in dichloromethane without using HOBt. In the case of coupling reactions with ordinary L-amino acid phenacyl esters, no racemization was observed. Examination of some model systems yielded sufficient evidence to prove that HOBt is an efficient catalyst for racemizing proline or hydroxy-proline phenacyl ester not only in the stage of cyclic intermediate formation but also in the opening of the ring structure. Thus, the racemization reaction was found to be closely related to the formation of the cyclic carbinol-amine derivative.     

Limiting racemization and aspartimide formation in microwave-enhanced Fmoc solid phase peptide synthesis.

Microwave energy represents an efficient manner to accelerate both the deprotection and coupling reactions in 9-fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS). Typical SPPS side reactions including racemization and aspartimide formation can occur with microwave energy but can easily be controlled by routine use of optimized methods. Cysteine, histidine, and aspartic acid were susceptible to racemization during microwave SPPS of a model 20mer peptide containing all 20 natural amino acids. Lowering the microwave coupling temperature from 80 degrees C to 50 degrees C limited racemization of histidine and cysteine. Additionally, coupling of both histidine and cysteine can be performed conventionally while the rest of the peptide is synthesized using microwave without any deleterious effect, as racemization during the coupling reaction was limited to the activated ester state of the amino acids up to 80 degrees C. Use of the hindered amine, collidine, in the coupling reaction also minimized formation of D-cysteine. Aspartimide formation and subsequent racemization of aspartic acid was reduced by the addition of HOBt to the deprotection solution and/or use of piperazine in place of piperidine.     

Trans-sulfuration Pathway Seleno-amino Acids Are Mediators of Selenomethionine Toxicity in Saccharomyces cerevisiae*

Toxicity of selenomethionine, an organic derivative of selenium widely used as supplement in human diets, was studied in the model organism Saccharomyces cerevisiae. Several DNA repair-deficient strains hypersensitive to selenide displayed wild-type growth rate properties in the presence of selenomethionine indicating that selenide and selenomethionine exert their toxicity via distinct mechanisms. Cytotoxicity of selenomethionine decreased when the extracellular concentration of methionine or S-adenosylmethionine was increased. This protection resulted from competition between the S- and Se-compounds along the downstream metabolic pathways inside the cell. By comparing the sensitivity to selenomethionine of mutants impaired in the sulfur amino acid pathway, we excluded a toxic effect of Se-adenosylmethionine, Se-adenosylhomocysteine, or of any compound in the methionine salvage pathway. Instead, we found that selenomethionine toxicity is mediated by the trans-sulfuration pathway amino acids selenohomocysteine and/or selenocysteine. Involvement of superoxide radicals in selenomethionine toxicity in vivo is suggested by the hypersensitivity of a Δsod1 mutant strain, increased resistance afforded by the superoxide scavenger manganese, and inactivation of aconitase. In parallel, we showed that, in vitro, the complete oxidation of the selenol function of selenocysteine or selenohomocysteine by dioxygen is achieved within a few minutes at neutral pH and produces superoxide radicals. These results establish a link between superoxide production and trans-sulfuration pathway seleno-amino acids and emphasize the importance of the selenol function in the mechanism of organic selenium toxicity.