Oxidative stress contributes to the pathophysiology of a variety of diseases, and circulating biomarkers of its severity remains a topic of great interest for researchers. Our peptidomic strategy enables accurate and reproducible analysis of circulating proteins/peptides with or without post-translational modifications. Conventional wisdom holds that hydrophobic methionines exposed to an aqueous environment or experimental handling procedures are vulnerable to oxidation. However, we show that the mass spectra intensity ratio of oxidized to non-oxidized methionine residues in serum tryptic proteins can be accurately quantified using a single drop of human serum and give stable and reproducible results. Our data demonstrate that two methionine residues in serum albumin (Met-111 and Met-147) are highly oxidized to methionine sulfoxide in patients with diabetes and renal failure and in healthy smokers versus non-smoker controls. This label-free mass spectrometry approach to quantify redox changes in methionine residues should facilitate the identification of additional circulating biomarkers suitable for predicting the development or progression of human diseases.

Oxidative stress has been linked to a number of chronic diseases, and this has aroused interest in the identification of clinical biomarkers that can accurately assess its severity. We used liquid chromatography-high resolution mass spectrometry (LC-MS) to show that oxidised and non-oxidised Met residues at position 147 of human serum albumin (Met 147) can be accurately and reproducibly quantified with stable isotope-labelled peptides. Met 147 oxidation was significantly higher in patients with diabetes than in controls. Least square multivariate analysis revealed that glycated haemoglobin (HbA 1c) and glycated albumin (GA) did not significantly influence Met 147 oxidation, but the GA/HbA 1c ratio, which reflects glycaemic excursions, independently affected Met 147 oxidation status. Continuous glucose monitoring revealed that Met 147 oxidation strongly correlates with the standard deviation of sensor glucose concentrations and the time spent with hypoglycaemia or hyperglycaemia each day. Thus, glycaemic variability and hypoglycaemia in diabetes may be associated with greater oxidation of Met 147. Renal function, high-density lipoprotein-cholesterol and serum bilirubin were also associated with the oxidation status of Met 147. In conclusion, the quantification of oxidised and non-oxidised Met 147 in serum albumin using our LC-MS methodology could be used to assess the degree of intravascular oxidative stress induced by hypoglycaemia and glycaemic fluctuations in diabetes. Oxidative stress is involved in a number of disease processes, including cardiovascular diseases 1,2 , diabetes 3-7 , chronic kidney disease 8-10 , cancer 11,12 , hypertension 2 and neurodegenerative disorders 13,14. Oxidative stress is also believed to be associated with ageing-associated disorders 15,16. Functional oxidative modification of biomolecules, including intravascular and cellular proteins, may have a causal role in the cellular dysfunctions that are involved in disease pathophysiology 17,18. The identification of clinical biomarkers of the severity of exposure to oxidative stress has been the intense focus of many researchers 19,20 , because they could be used to predict the development of major human diseases. Because the quantification of reactive oxygen species is difficult, given their very short half-lives, the measurement of stable by-products generated under conditions of oxidative stress remains a popular approach to the monitoring of free radical-influenced processes 20. Methionine (Met), a sulfur-containing amino acid, is an important antioxidant that contributes to the structure and stability of proteins 21. Met is readily oxidised to form Met sulfoxide (MetO), which can be reduced back to Met by MetO reductases 22-26. Because of this instability of Met and MetO, their quantification has not been a widely used method for the assessment of the degree of oxidative stress 27. However, we have recently found that the mass spectral intensity of serum tryptic peptides containing oxidised and non-oxidised Met residues can be very...

There is a high risk of asymptomatic hypoglycemia associated with hemodialysis (HD) using glucose-free dialysate; therefore, the inclusion of glucose in the dialysate is believed to prevent intradialytic hypoglycemia. However, the exact glycemic fluctuation profiles and frequency of asymptomatic hypoglycemia using dialysates containing >100 mg/dL glucose have not been determined. RESEARCH DESIGN AND METHODSWe evaluated the glycemic profiles of 98 patients, 68 of whom were men, with type 2 diabetes undergoing HD (HbA 1c 6.4 ± 1.2%; glycated albumin 20.8 ± 6.8%) with a dialysate containing 100, 125, or 150 mg/dL glucose using continuous glucose monitoring. RESULTSSensor glucose level (SGL) showed a sustained decrease during HD, irrespective of the dialysate glucose concentration, and reached a nadir that was lower than the dialysate glucose concentration in 49 participants (50%). Twenty-one participants (21%) presented with HD-related hypoglycemia, defined by an SGL <70 mg/dL during HD and/or between the end of HD and their next meal. All these hypoglycemic episodes were asymptomatic. Measures of glycemic variability calculated using the SGL data (SD, coefficient of variation, and range of SGL) were higher and time below range (<70 mg/dL) was lower in participants who experienced HD-related hypoglycemia than in those who did not, whereas time in range between 70 and 180 mg/dL, time above range (>180 mg/dL), HbA 1c , and glycated albumin of the two groups were similar. CONCLUSIONSDespite the use of dialysate containing 100-150 mg/dL glucose, patients with diabetes undergoing HD experienced HD-related hypoglycemia unawareness frequently. SGL may fall well below the dialysate glucose concentration toward the end of HD.Diabetes is a major cause of end-stage kidney disease and cardiovascular disease (1,2). The prognosis of patients with diabetes undergoing maintenance hemodialysis (HD) is worse than that of patients without diabetes undergoing HD (3), but it remains unclear whether differences in glycemic profile affect the high mortality

Glucose-dependent insulinotropic polypeptide (GIP), secreted from enteroendocrine K cells, has potent insulinreleasing and extrapancreatic glucoregulatory activities. However, exogenous GIP has less potent biological effects compared with another incretin hormone, GLP-1, which limits its use for the treatment of type 2 diabetes. The fate and secretion of administered native GIP remain unclear. The aim of this study was to identify plasma binding proteins for human GIP. Fluorescent-labelled GIP was added to fresh human plasma and subjected to clear native polyacrylamide gel electrophoresis (CN-PAGE). Then fluorescent protein bands were in-gel trypsin-digested and subjected to liquid chromatography tandemmass spectrometry (LC-MS/MS) analysis, revealing the presence of albumin, immunoglobulin G (IgG) and transferrin. In contrast to GIP, the binding of fluorescent GLP-1 and glucagon to plasma protein fractions were minimal. CN-PAGE analysis of synthetic GIP incubated with human serum albumin, purified IgG or transferrin, and subsequent western blot analysis revealed that GIP binds to each of these proteins. Taken together, these results indicate that GIP readily binds to albumin, IgG and transferrin, three plasma proteins highly abundant in the human peripheral circulation. Separation of protein complexes using CN-PAGE and the identification of in-gel digested proteins by LC-MS/MS analysis provide a promising strategy to identify plasma binding proteins for bioactive peptides.

<b>OBJECTIVE</b> <p>There is a high risk of asymptomatic hypoglycemia associated with hemodialysis (HD) using glucose-free dialysate; therefore, the inclusion of glucose in the dialysate is believed to prevent intradialytic hypoglycemia. However, the exact glycemic fluctuation profiles and frequency of asymptomatic hypoglycemia using dialysates containing >100 mg/dL glucose have not been determined.</p> <p><b>RESEARCH DESIGN AND METHODS</b></p> <p>We evaluated the glycemic profiles of 98 type 2 diabetes patients undergoing HD (68 men, HbA1c 6.4±1.2%, glycated albumin 20.8±6.8%) with a dialysate containing 100, 125, or 150 mg/dL glucose using continuous glucose monitoring.</p> <p><b>RESULTS</b></p> <p>Sensor glucose level (SGL) showed a sustained decrease during HD, irrespective of the dialysate glucose concentration, and reached a nadir that was lower than the dialysate glucose concentration in 49 participants (50%). Twenty-one participants (21%) presented with HD-related hypoglycemia, defined by an SGL <70 mg/dL during HD and/or between the end of HD and their next meal. All these hypoglycemic episodes were asymptomatic. Measures of glycemic variability calculated using the SGL data (standard deviation, coefficient of variation, and range of SGL) were higher and time below range (<70 mg/dL) was lower in participants who experienced HD-related hypoglycemia than in those who did not, whereas time in range between 70 and 180 mg/dL, time above range (>180 mg/dL), HbA1c and GA of the two groups were similar.</p> <p><b>CONCLUSIONS</b></p> <p>Despite the use of dialysate containing 100–150 mg/dL glucose, diabetic HD patients experienced HD-related hypoglycemia unawareness frequently. SGL may fall well below the dialysate glucose concentration toward the end of HD.<br> </p>