Oxidised Met147 of human serum albumin is a biomarker of oxidative stress, reflecting glycaemic fluctuations and hypoglycaemia in diabetes

Abstract

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...

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“…2 ). The plasma concentrations were then extrapolated from the XICs generated using the respective endogenous peptides and the corresponding spiked stable isotope-labeled peptides as described 26 .…”

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