How calcium causes microtubule depolymerization

The effect of replacing bovine serum albumin (BSA) in a simple defined medium (KSOM) with polyvinyl alcohol (PVA) and/or amino acids on the percentages of mouse zygotes that develop to at least the blastocyst stage and that hatch at least partially or completely is reported. Blastocysts could form when BSA was replaced with only PVA, but at a moderately reduced rate; however, partial hatching, and hence complete hatching, were severely impaired when BSA was replaced with only PVA. The substitution of BSA with amino acids alone resulted in a high rate of blastocyst formation and moderate impairment of hatching. The addition of PVA to BSA-free KSOM supplemented with amino acids had no extra effect. BSA had significant effects when added to BSA-free KSOM supplemented with amino acids. The BSA caused a significant increase in the rate of partial hatching, and may even have had a small effect on the rate of blastocyst formation. The results also showed that glucose, at a high concentration of 5.56 mM, does not inhibit the development of mouse zygotes to hatched blastocysts when cultured in KSOM supplemented with amino acids.     

Differential regulation of mouse embryo development and viability by amino acids

The amino acid requirements of the preimplantation mouse embryo in culture changes as development proceeds from the zygote to the blastocyst stage. Eagle's non-essential amino acids and glutamine significantly increased cleavage rates during the first four cell cycles, while Eagle's essential amino acids without glutamine did not confer any benefit to embryo development before the eight-cell stage. After the eight-cell stage, non-essential amino acids and glutamine no longer stimulated cleavage rates but significantly increased blastocoel development and blastocyst hatching. In contrast, after the eight-cell stage essential amino acids increased cleavage rates as well as stimulating development of the inner cell mass of the resultant blastocysts. Fetal development after transfer of blastocysts was also significantly increased by culture with essential amino acids from the eight-cell stage. Consequently highest rates of development in vitro and viability after transfer were achieved when embryos were cultured with non-essential amino acids and glutamine to the eight-cell stage followed by development to the blastocyst stage in the presence of all 20 amino acids. Analysis of the parameters measured revealed a significant relationship between number of blastocyst cells and inner cell mass development with viability after transfer. Blastocyst formation and hatching could not be used to assess subsequent developmental potential.     

Evidence for the requirement of autocrine growth factors for development of mouse preimplantation embryos in vitro

The mitotic stimuli in the early mammalian embryo have not been unequivocally identified. One hypothesis is that the embryo releases autocrine growth factors (GFs) that have a role in such growth. To determine whether such putative GFs were limited by dilution, and hence secreted, development was observed at various embryo concentrations in culture. Embryos were collected at the zygote or 2-cell stage. Zygotes were produced by fertilization in situ (ISF) or in vitro (IVF). Two-cell-stage embryos had a high rate of development to the blastocyst stage across an embryo concentration range of 1/microl-0.001/microl. By contrast, zygotes produced by either ISF or IVF were adversely affected by reducing the embryo concentration over this range (p < 0.001), with approximately 80% of ISF zygotes developing to blastocysts at the highest concentration but only 26% at the lowest. For IVF zygotes the corresponding results were 64% and 6%. For all three embryo types, the number of cells in each blastocyst was significantly lower with reduced embryo concentration. The major determinant of zygote development was the concentration of embryos in culture rather than the absolute volume of culture medium or the actual number of embryos present. A concentration of 1 embryo/microl (in the form of 10 embryos/10microl) gave the best development rates and highest cell numbers per blastocyst. Varying the albumin concentration influenced development rates; a 10-fold reduction in BSA concentration (to 0.3 mg/ml) resulted in significantly more IVF zygotes developing to the blastocyst stage. Platelet-activating factor (PAF) is released by embryos, and albumin can act as a competitive inhibitor of PAF's action on cells. ISF embryos released more PAF (p < 0.05) into media than did similarly treated IVF embryos. There was no difference in the amount of PAF remaining associated with the resulting 2-cell embryos. The amount of PAF released by both these groups was markedly less (p < 0.001) than the amount released by 2-cell embryos collected fresh from the reproductive tract and cultured for 24 h. PAF supplementation of media caused a significant increase in the rate of blastocyst development of IVF zygotes at embryo concentrations of 0.1/microl (1 ng/ml) and 0.01/microl (100 ng/ml). Insulin-like growth factor (IGF)-I (30 ng/ml) and IGF-II (1 ng/ml) also stimulated development of IVF zygotes when cultured at an embryo concentration of 1/10 microl. Epidermal growth factor was without effect over the range 0.2-2000 ng/ml. Supplementation of media with both PAF and IGF-II gave no additional benefit over that caused by IGF-II alone, but this treatment was marginally better (p < 0.05) than PAF treatment alone. The results show that factors necessary for normal embryo development are diluted to suboptimal levels during culture at low embryo concentration. The ability of PAF, IGF-I, and IGF-II to partially compensate for the adverse effects of low embryo concentration during culture is consistent with their having roles as autocrine embryotrophic factors. The use of IVF and low embryo concentrations in culture may provide a functional multiple ablation model that will help to define the range of GFs required for normal embryo development.     

Brief family therapy: an option for the treatment of somatoform disorders in primary care

Using defatted and SH-blocked bovine serum albumin (BSA), measurements of differential scanning calorimetry (DSC) have been made at pH 7 on the complexes of BSA and a series of sodium alkyl sulfates used were sodium decyl sulfate (SDeS), sodium octyl sulfate (SOS), sodium hexyl sulfate (SHS) and sodium ethyl sulfate (SES). Results obtained were compared with those on the system BSA-sodium dodecyl sulfate (SDS) studied previously. Two peaks P1 and P2 existed in the DSC curve of BSA. These peaks originate in the heat-induced transition of BSA. The pattern of DSC curve changed with the amount of the ligand added, i.e. with the molar mixing ratio ligand/BSA (1). The change for systems BSA-SDeS, BSA-SOS and BSA-SHS was qualitatively the same as that for the system BSA-SDS (2). Interestingly, SES, which is not a surfactant, interacts with BSA. The change for the system BSA-SES was qualitatively the same as that for the system BSA-Na2SO4. All alkyl sulfates suppressed the heat-induced transition at lower concentrations. A linear relationship was obtained for the plots of log(D/A)1 versus log CMC, where (D/A)1 is the molar mixing ratio of anionic surfactant (D) to BSA (A) at which the most heat-stable complex is formed. This suggests that the hydrophobic force has a serious effect on the formation of heat-stable complexes.     

Effect of certain diuretics on thyroid function in rats

The effects of isolated protein fractions from rabbit uteri (prealbumin, albumin, uteroglobin, and beta-glycoprotein), unfractionated uterine proteins, progesterone, oestradiol-17beta, and prostaglandin F-2a on the development of rabbit embryos in vitro were investigated. When exposed to individual protein fractions obtained from Day-6 uteri, 8-cell embryos did not develop into early blastocysts; morulae readily developed into early blastocysts, but further development was retarded. Progesterone (10(-5)-10(-11)M) and prostaglandin F-2a (0-1-10 ng/ml) added to the medium slowed development of blastocysts to advanced stages. Growth of 8- to 16-cell embryos, morulae, and Day-4 blastocysts was stimulated by unfractionated uterine proteins obtained from Day-5 uterine flushings. Although embryos cultured in medium containing BSA had similar rates of blastocyst formation and, ultimately similar blastocyst expansion as did the embryos cultured in medium with unfractionated proteins, the radial and immediate expansion of the early blastocysts cultured in the latter approximated that found in utero.     

Mechanism of the growth-promoting effect of serum albumin on concanavalin A-activated lymphocytes: protective effect of the plasma proteins

The concentration of concanavalin A (Con A) required for optimal growth of lymphocytes is 10 to 20 times greater in the presence of plasma than in the presence of serum albumin (BSA). This shift in Con A requirement is mainly caused by Cohn fractions III and IV, which bind and probably remove free Con A. Aymphocytes, once they are activated by Con A, lose all proliferative activity unless protected by certain proteins. Cohn fraction VI and beta-lactoglobulin were found to be most effective in protecting the cells. The protective proteins do not promote growth; only BSA promotes growth of Con A-activated lymphocytes. The mechanism of the growth-promoting effect of BSA on Con A-activated lymphocytes was investigated. The continous presence of BSA in the culture medium durdly reduces, whereas protein-free medium abolishes, growth. The mechanism of the growth-promoting effect of BSA is not concerned with regulating the uptake of Con Aby lymphocytes. Red blood cells, crenated by washing in protein-free medium, revert immediately to their normal globular shape by the addition ofBSA or FAFBSA, whereas the protective proteins fail to do so, indicating a direct effect of BSA on the external surface of the erythrocyte membrane and presumably also of the lymphocyte. The hypothesis is proposed that BSA promotes growth by conveting total structural integrity to the cell surface membrane of Con A-treated lymphocytes.     

Enhanced immune response after subcutaneous and oral immunization with biodegradable PLGA microspheres

PLGA microspheres containing bovine serum albumin (BSA) as a model antigen, were prepared by a double emulsion/solvent extraction method and their in vitro characterization was performed. The same microspheres were used in a series of in vivo studies to evaluate the immune response induced after subcutaneous or oral inoculation following different immunization protocols. The in vivo data confirm that the immunogenicity of the albumin is not affected by the encapsulation procedure. The subcutaneous administration of microspheres showed an immune response (serum IgG levels by ELISA) statistically above BSA solution, even when the dose administered was 10 times lower. The adjuvanticity of the microspheres was found to be comparable to that of Freund's complete adjuvant (FCA), but in contrast to FCA they are biocompatible and did not induce any adverse reaction at the site of injection. A single oral administration of the microspheres was not a successful strategy for the induction of a reproducible response. Therefore, microspheres of 1 and 5 micrometer were orally administered on 3 consecutive days and the response obtained showed that the use of a boosting dose was not necessary for the 1 micrometer particles. These results suggest the possibility of simplifying the immunization schedule to a primary immunization if 1 micrometer particles are administered.     

Formaldehyde in cryoprotectant propanediol and effect on mouse zygotes

Cryopreservation of human zygotes and embryos has been routinely performed by in-vitro fertilization clinics for many years. Karran and Legge (1996) first reported that formaldehyde (FA) present in the cryoprotective solutions can have a deleterious effect on mouse oocytes. FA is a cytotoxic, carcinogenic and mutagenic chemical. The effect of FA on mouse zygotes was investigated. In addition, the concentrations of FA in propanediol (PROH) obtained from various sources were determined. Pooled 1-cell embryos were dispensed into droplets of modified Ham's F10 or human tubal fluid containing various concentrations of FA. Since bovine serum albumin (BSA) may minimize toxicity additional trials were done as above in the absence of BSA. FA concentration in the standard 1.5 M PROH, from different sources in water, was measured in the same assay using a standard curve of 0-100 microM FA. FA in a complex medium had a significant deleterious effect on embryo development and hatching but only at 1 mM concentration (P < 0.000001; see Tables I-III). There was no significant effect of FA at 100 microM. However, in a simple medium even 50 microM FA decreased embryo hatching. FA was present in 1.5 M PROH from different sources (range 1.0-35.3 microM concentration). It appears that FA concentrations do not increase with storage because FA concentrations were low even after opening and storage for 3 years on the shelf. This suggests that FA is a contaminant during the manufacturing process and may vary from manufacturer to manufacturer and batch to batch. Until further studies are done to confirm the lack of toxicity to embryos during cryopreservation (with or without FA scavengers) it may be prudent to screen all batches of cryoprotectants for FA as part of quality control.     

Action of bovine serum albumin on cytochrome c oxidase activity and proton pumping: a role for fatty acids in enzyme function?

Bovine serum albumin (BSA) at micromolar concentrations causes a red shift of the Soret band of bovine cytochrome c oxidase with a slow biphasic time course. It also inhibits the turnover of detergent-isolated enzyme in a similarly slow manner; the progress of this inhibition is halted by palmitate and other fatty acids. The inhibitory bovine serum albumin effect may involve fatty acid depletion from the enzyme. Respiration by cytochrome c oxidase vesicles (proteoliposomes) in the presence of ionophores (uncontrolled) shows only a small inhibition by BSA but preincubation of such vesicles with BSA induces a loss of proton pumping activity. After incubation of BSA-depleted proteoliposomes in the presence of reductant with combinations of fatty acids, pumping activity can be fully restored, suggesting a supportive or even essential role of endogenous fatty acids in H+ translocation by this membranous enzyme.     

Activity of glutathione-dependent enzymes in long term diabetes. II. Glutathione contents and activity of glutathione-dependent enzymes: S-transferase and peroxidase in the kidney cytosol of alloxan induced diabetic rats

To better understand the uptake of oligonucleotides into cells, we have studied the labeling of cell surface proteins by an oligonucleotide conjugated to a radiolabeled photoactivatable crosslinker (Denny-Jaffe reagent). When HL60 cells are treated with the conjugate for 2 hours in a medium containing bovine serum albumin (BSA), almost all of the cell-associated label is found in one protein, which we identify as BSA. Cells grown and treated in a serum-free medium do not show this protein, whereas it is plainly seen in cells that are grown in serum-containing medium but then treated in serum-free medium. Overall association of the oligonucleotide with cells is much higher in serum-free medium than in BSA-containing medium, but the oligonucleotide is mostly not protein-associated in the absence of BSA. We conclude that (1) BSA from the medium serves to block overall association of oligonucleotide with cells, and (2) BSA is the main cell surface protein binding oligonucleotides. We discuss the possible role of albumin in endocytic uptake of oligonucleotides in the cell and in the biodistribution of oligonucleotides in vivo.     

Influence of adjuvants on the induction of autoantibodies to the thyrotropin receptor

To determine the influence of adjuvant on the induction of antibodies to thyrotropin receptor (TSHR), we immunized BALB/c mice with a extracellular domain of the TSHR (ETSHR) protein in complete Freund's adjuvant (CFA), Titer Max (TM) and Gerbu. Similarly, control groups of mice were immunized with bovine serum albumin (BSA) in each of the different adjuvants. As determined by ELISA, ETSHR given along with CFA elicited high titers of antibodies to ETSHR which were mainly restricted to the IgG1 subclass. Mice immunized with ETSHR in TM also developed high titers of anti-ETSHR antibodies but had higher levels of both IgG1 and IgG2a. However, immunization with ETSHR in Gerbu resulted in low titers of antibodies, restricted to IgG1 subclass. Immunization of mice with BSA in each of the three adjuvants induced higher antibody titers to BSA. The subclass of antibodies in mice immunized with BSA in CFA and TM were predominantly IgG1 and IgG2a with lower levels of IgG2b, whereas in Gerbu treated group, antibody to BSA was restricted to IgG1 subclass. Analysis of specificity of antibodies against ETSHR, in mice immunized with ETSHR, revealed that irrespective of the adjuvant used, the dominant reactivity was against peptide 1 (AA 22-41) with weaker reactivity against several other. peptides. The only exception was in mice immunized with ETSHR in TM which also showed significant reactivity against peptide 23 (AA 352-371). Mice immunized with the ETSHR in CFA or in TM showed elevated levels of serum TSH binding inhibitory immunoglobulins (TBII). However, mice immunized with ETSHR in Gerbu, which had lower titers of antibodies to ETSHR, showed normal TBII levels. These studies showed that adjuvant composition could influence the titer, subclass and fine specificity of antibodies to ETSHR which in turn could affect the development of TBII activity.     

Half a century of experience in the experimental and clinical study of virus infections]

An analysis of farm workers and rural dwellers for serum antibodies to the lipopolysaccharide (LPS) of Escherichia coli O157 detected sera with antibodies binding to bovine serum albumin (BSA) by ELISA. These antibodies were not specific for BSA when examined by immunoblotting, and the ELISA values were reduced to a background level when plates were blocked with normal rabbit serum.     

Smoking cessation in pregnancy. Intervention among heavy smokers

OBJECTIVE: Human recombinant leukemia inhibitory factor (rLIF) has been shown to stimulate hatching of murine and ovine embryos in vitro. The temporal and dose-dependent effects of murine rLIF (mrLIF) and human rLIF (hrLIF) on embryo development in two different mouse strains were investigated in this work. METHODS: Two-cell embryos were recovered from the fallopian tubes of superovulated/mated females and cultured in Krebs medium plus bovine serum albumin in microdroplets under oil. RESULTS: In the B6CBF1 strain, mrLIF significantly stimulated blastocyst formation and decreased embryo fragmentation/degeneration when added simultaneously at the initiation of culture or 24 hours thereafter. Human rLIF also had a positive effect on development. In the CD1 strain (lower fecundity), mrLIF dose-dependent effects were observed, with enhanced developmental stimulation achieved with higher doses. CONCLUSIONS: These findings confirm that hrLIF stimulates mouse embryo development in vitro and that different mouse strains show distinct responses to the cytokine. In addition, mrLIF enhances blastocyst formation and decreases embryo fragmentation when added to the embryo culture as early as the two-cell stage.     

Constuction of an improved system for the determination of fidelity of polymerase in PCR

Indole-3-acetic acid (IAA) is a product of tryptophan (Trp) metabolism and is found to be markedly increased in uremic sera. IAA binding to defatted human serum albumin at 37 degrees C and pH 5, 7.4, and 8.5 was studied by equilibrium dialysis, and data were analyzed assuming two independent high affinity binding sites plus a class of low affinity sites. The estimated values of the association constant of dominant site were: 7.96 x 10(3) M-1 at pH 5, 11.57 x 10(3) M-1 at pH 7.4, and 6.30 x 10(3) M-1 at pH 8.5. The competition between IAA and Trp for albumin binding at pH 7.4 was investigated. The results suggest that one specific albumin site is common for IAA and Trp, but the data were not adequately predicted by a purely competitive scheme. A better prediction was achieved assuming that the binding of IAA to a site different from the common site inhibits Trp binding.     

Growth and survival of Helicobacter pylori in defined medium and susceptibility to Brij 78

The gastrointestinal pathogen Helicobacter pylori requires supplementation with either fetal calf serum (FCS), bovine serum albumin (BSA), or (2,6-dimethyl)-beta-cyclodextrin (CD) for growth in a complex or defined medium. Because the availability of medium in which all components were chemically defined would facilitate metabolic studies of H. pylori, growth of the type strain, ATCC 43504, was compared in a defined medium with different growth additives. The dependency of H. pylori growth on FCS or BSA in a defined medium could partially be replaced by dependency on CD and cholesterol when the last two components were both added to the defined medium. Growth and cell yield were not affected by the addition of glucose, but the culture viability (numbers of CFU per milliliter was extended. Because therapeutic antifoams are used to relieve gastrointestinal symptoms we studied whether the unique susceptibility of H. pylori to the emulsifier polyoxyethylene-20-stearylether (Brij 78) was growth dependent or medium specific. The bactericidal activity exerted in buffer at pH 5 was independent of the preculture medium, and a 5-h exposure of the bacteria to 1.28 to 2.56 microg of Brij 78 per ml reduced the numbers of viable bacteria by >5 log10. The MICs (0.16 to 0.32 microg/ml) were lower than the corresponding minimal bactericidal concentrations in different growth media and were affected by FCS or BSA. In conclusion, CD plus cholesterol promotes the growth of H. pylori in a serum-free defined medium in which glucose enhances cell viability. The antibacterial activity exerted by Brij 78 is neither growth dependent nor medium specific.     

Advanced glycated albumin impairs protein degradation in the kidney proximal tubules cell line LLC-PK1

Advanced glycation end-products (AGEs) are assumed to play a major role in the genesis of diabetic nephropathy and other diabetic complications. We studied the potential effect of AGEs on protein turnover and lysosomal proteinase activities in LLC-PK1 cells, a pig kidney proximal tubules cell line. Advanced glycated bovine serum albumin (AGE-BSA) was used as a model of AGEs and its action was compared to that of nonglycated BSA. AGE-BSA but not BSA (50 micromol/l) induced a significant increase in cell volume (BSA: 4870.6 +/- 74.8 fl, AGE-BSA: 5718.0 +/- 20.7 fl, p<0.01). Protein degradation rate was decreased by 13.8% after 48 hrs. incubation with AGE-BSA (p<0.01) while protein synthesis increased by 19,1%, (p<0.01). After incubation with AGE-BSA but not BSA activities of lysosomal cathepsins (B, L+B and H) decreased in a time- and dose-dependent fashion. This decline was neither caused by a shift in lysosomal pH outside the optimal range for cathepsins, nor by a direct inhibitory effect of AGEs modified proteins or peptides but most probably by inhibition of cathepsin B expression as measured by RT-PCR. It is supposed that impaired protease activities participated in decreased protein breakdown and cell enlargement. For the first time our data provide the evidence that AGEs induce hypertrophy of LLC-PK1 cells due to decreased protein breakdown resulting from reduced lysosomal proteinase activities with a concomitant stimulation of protein synthesis.     

Characterization of the embryotrophic activity of exogenous protein-free oviduct-conditioned medium used in culture of cattle embryos

Bovine zygotes, obtained after in vitro maturation and fertilization of oocytes from slaughtered cow ovaries, were cultured in droplets of nonconditioned or conditioned medium on bovine oviduct cell monolayers. The media tested were Medium 199 alone and Medium 199 supplemented with 10% fetal calf serum (FCS). Oviduct conditioning increased both early cleavage and development to blastocysts. Only the effect on early cleavage was mimicked by FCS. The blastocysts obtained in serum-free conditioned medium (SFCM) appeared morphologically normal and had the same cell number as those produced in conditioned medium containing serum. Their hatching rates did not differ. Transfer of 16 blastocysts developed in SFCM to 16 synchronized recipients resulted in five pregnancies (31%), indicating good embryonal viability. Boiling of SFCM resulted in a total loss of activity, while heating at 56 degrees C for 30 min had no deleterious effect. A 10-kDa ultrafiltration of SFCM removed the blastocyst development-supporting activity from the filtrate but not the early cleavage-favoring activity. This allows us to conclude that at least two different factors are present in SFCM: one of low molecular mass (< approximately 10 kDa), needed to obtain the 5-8 cell stage and mimicked by FCS, and another of higher molecular mass allowing embryos to develop from the 8-cell to blastocyst stage.     

Trophic effects of myeloid leukaemia inhibitory factor (LIF) on mouse embryos

Myeloid leukaemia inhibitory factor (LIF) is expressed at highest concentrations in the maternal endometrial glands at about the stage of blastocyst implantation. LIF is also expressed by the extraembryonic membranes of the early mouse embryo. Embryos of different ages were cultured with, or without, LIF, and embryo growth in vivo and in vitro was examined to determine whether LIF is important for embryo development. Supplementing embryo culture media with 1000 U recombinant human LIF ml-1 increased the number of eight-cell mouse embryos developing beyond the hatched blastocyst stage in vitro from 62.1% to 85.1% (P < 0.05). LIF significantly increased the number of embryos hatching (33.8% versus 7.65% for controls 96 h after hCG injection, P < 0.001), completely hatching (85.1% versus 62.1%, P < 0.05), and exhibiting trophoblast outgrowth (13.5% versus 0% 120 h after hCG treatment, 85.1% versus 47.0% 144 h after hCG treatment, P < 0.001) in vitro. LIF-treated embryos also displayed a significantly greater area of trophoblast outgrowth than did controls as early as day 5 in culture (P < 0.005). These data show that LIF enhances mouse eight-cell embryo development in vitro, as seen by the accelerated rate of embryo hatching and trophoblast outgrowth. In addition, enhanced embryo survival in vivo is shown, following the transfer of LIF-treated embryos into a pseudopregnant recipient female. Expression of mRNA encoding LIF was detected in endometrial cells cultured in monolayer from uteri of day 3 pregnant females, explaining the known embryotrophic effects of endometrial coculture. This expression was not enhanced significantly by treatment with oestradiol (3.7 x 10(-5) mol l-1) or progesterone (3.2 x 10(-6) mol l-1) or both hormones. These results indicate that LIF could have a dual action in early embryogenesis as an embryotrophin and as a factor required for embryo implantation. Multiple roles for LIF are consistent with the expression of this factor at embryonic, extraembryonic and maternal sites during early embryogenesis.     

Glucose and free radicals impair the antioxidant properties of serum albumin

Epidemiological data consistently show that reduced levels of serum albumin, which is the most abundant protein in plasma, are associated with an increased mortality risk. Various biological properties evidenced by direct effects of the albumin molecule may explain its beneficial effects. The present work aimed to investigate in vitro whether glycation or free radicals or both factors would affect the antioxidant properties of bovine serum albumin (BSA). Glycation was performed by long-term incubations (60 days) of BSA with increasing concentrations of glucose (up to 500 mmol/l) at 37 degreesC. Minimally oxidized BSA was obtained after controlled incubations of dialyzed BSA samples with a water-soluble free radical generator [2,2' azo-bis(2-amidinopropane) HCl]. The glycation-mediated modifications and the free radical-induced conformational changes of BSA were monitored using intrinsic fluorescence measurements of the tryptophan residues and acrylamide as a quenching agent. Thiol groups, Amadori glycophore contents, and boronate binding were also measured. We found that the changes observed in the conformation of the BSA molecule were associated with modifications of its antioxidant properties. The latter were studied by the copper-mediated oxidation of human low density lipoproteins and the free radical-induced blood hemolysis test. Our data support the concept that oxidative-induced BSA modifications are important determinants in the antioxidant properties of BSA. Glycated BSA still behaved as an antioxidant but became pro-oxidant in the presence of copper, probably by generating oxygenated species. These data confirm the key role of metals ions in this process. Although these results warrant further in vivo investigations, we propose that, considering the poor glucose control found in diabetics as well as the key role of oxidative stress in vascular complications, glycation-mediated and free radical-induced impairment of the antioxidant properties of albumin might be important parameters in vascular complications encountered in diabetes.