Site specificity of glycation and carboxymethylation of bovine serum albumin by fructose

We report an investigation of the site specificity, extent and nature of modification of bovine serum albumin (BSA) incubated with fructose or glucose at physiological temperature and pH. Sites of early glycation (Heyns rearrangement products (HRP) from fructose; fructoselysine (FL) from glucose) as well as advanced glycation (N-epsilon-(carboxymethyl)lysine; CML) wereanalyzed by liquid chromatography-mass spectrometry. The major site of modification by fructose, like glucose, is Lysine-524 and this results in, respectively, 31 and 76% loss of the corresponding unmodified tryptic peptide, Gln525-Lys533. In addition, total lysine, HRP, FL, CML and N-epsilon-(carboxyethyl)lysine in the incubations, was quantified. Almost all of the loss of lysine in the fructose-modified BSA was attributed to the formation of CML, with the yield of CML being up to 17-fold higher than glucose-modified BSA. A mechanism for the formation of CML from the HRP is proposed.

Early alterations in serum creatine kinase and total cholesterol following high intensity eccentric muscle actions

Aim. The purpose of this experiment was to assess the levels of muscle soreness, serum total cholesterol (TC) and creatine kinase (CK) in the first 48 hours following fatiguing eccentric exercise performed with the triceps brachii.

Elevated serum phosphate predicts mortality in renal transplant recipients

BACKGROUND.: High serum phosphate has been identified as an important contributor to the vascular calcification seen in patients with chronic kidney disease (Block et al., Am J Kidney Dis 1998; 31: 607). In patients on hemodialysis, elevated serum phosphate levels are an independent predictor of mortality (Block et al., Am J Kidney Dis 1998; 31: 607; Block, Curr Opin Nephrol Hypertens 2001; 10: 741). The aim of this study was to investigate whether an elevated serum phosphate level was an independent predictor of mortality in patients with a renal transplant.
METHODS.: Three hundred seventy-nine asymptomatic renal transplant recipients were recruited between June 2000 and December 2002. Serum phosphate was measured at baseline and prospective follow-up data were collected at a median of 2441 days after enrolment.
RESULTS.: Serum phosphate was significantly higher in those renal transplant recipients who died at follow-up when compared with those who were still alive at follow-up (P<0.001). In Kaplan-Meier analysis, serum phosphate concentration was a significant predictor of mortality (P=0.0001). In multivariate Cox regression analysis, serum phosphate concentration remained a statistically significant predictor of all-cause mortality after adjustment for traditional cardiovascular risk factors, estimated glomerular filtration rate, and high sensitivity C reactive protein (P=0.036) and after adjustment for renal graft failure (P=0.001).
CONCLUSIONS.: The results of this prospective study are the first to show that a higher serum phosphate is a predictor of mortality in patients with a renal transplant and suggest that serum phosphate provides additional, independent, prognostic information to that provided by traditional risk factors in the risk assessment of patients with a renal transplant.

Selection of an analytical method for evaluating bovine serum albumin concentrations in pharmaceutical polymeric formulations

Bovine serum albumin (BSA) is a commonly used model protein in the development of pharmaceutical formulations. In order to assay its release from various dosage forms, either the bicinchoninic acid (BCA) assay or a more specific size-exclusion high performance liquid chromatography (SE-HPLC) method are commonly employed. However, these can give erroneous results in the presence of some commonly-used pharmaceutical excipients. We therefore investigated the ability of these methods to accurately determine BSA concentrations in pharmaceutical formulations that also contained various polymers and compared them with a new and compared with a new reverse-phase (RP)–HPLC technique. We found that the RP-HPLC technique was the most suitable method. It gave a linear response in the range of 0.5 -100 µg/ml with a correlation coefficient of 0.9999, a limit of detection of 0.11 µg/ml and quantification of 0.33 µg/ml. The performed ‘t’ test for the estimated and theoretical concentration indicated no significant difference between them providing the accuracy. Low % relative standard deviation values (0.8-1.39%) indicate the precision of the method. Furthermore, the method was used to quantify in vitro BSA release from polymeric freeze-dried formulations.

Serum Antibodies to Porphyromonas gingivalis Chaperone HtpG Predict Health in Periodontitis Susceptible Patients

Chaperones are ubiquitous conserved proteins critical in stabilization of new proteins, repair/removal of defective proteins and immunodominant antigens in innate and adaptive immunity. Periodontal disease is a chronic inflammatory infection associated with infection by Porphyromonas gingivalis that culminates in the destruction of the supporting structures of the teeth. We previously reported studies of serum antibodies reactive with the human chaperone Hsp90 in gingivitis, a reversible form of gingival disease confined to the oral soft tissues. In those studies, antibodies were at their highest levels in subjects with the best oral health. We hypothesized that antibodies to the HSP90 homologue of P. gingivalis (HtpG) might be associated with protection/resistance against destructive periodontitis.

Upregulation of Oxidative Stress Markers in Human Microvascular Endothelial Cells by Complexes of Serum Albumin and Digestion Products of Glycated Casein

The extent of absorption of dietary advanced glycation end products (AGEs) is not fully known. The possible physiological impact of these absorbed components on inflammatory processes has been studied little and was the aim of this investigation. Aqueous solutions of bovine casein and glucose were heated at 95 degrees C for 5 h to give AGE-casein (AGE-Cas). Simulated stomach and small intestine digestion of AGE-Cas and dialysis (molecular mass cutoff of membrane = 1 kDa) resulted in a low molecular mass (LMM) fraction of digestion products, which was used to prepare bovine serum albumin (BSA)-LMM-AGE-Cas complexes. Stimulation of human microvascular endothelial cells with BSA-LMM-AGE-Cas complexes significantly increased mRNA expression of the receptor of AGE (RAGE), galectin-3 (AGE-113), tumor necrosis factor alpha, and a marker of the mitogen-activated protein kinase pathway (MAPK-1), as well as p65NF-kappa B activation. Cells treated with LMM digestion products of AGE-Cas significantly increased AGE-R3 mRNA expression. Intracellular reactive oxygen species production increased significantly in cells challenged with BSA-LMM-AGE-Cas and LMM-AGE-Cas. In conclusion, in an in vitro cell system, digested dietary AGEs complexed with serum albumin play a role in the regulation of RAGE and down-stream inflammatory pathways. AGE-R3 may protect against these effects.