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.
Rabbit alveolar macrophage microsomes were found to acylate 1 -[ 'HI alkylglycero-3phosphocholine (GPC) (lyso platelet-activating factor) in the absence of any cofactors, indicating the presence of transacylation activity.The transacylation activity was comparable to the activity of acyl-CoA:l-alkyl-GPC acyltransferase.The fatty acyl moieties introduced into 1- ['Hlalkyl-GPC from membrane lipids by microsomes were mainly 20:4 (n -6).A very similar acylation profile was observed for the acylation of l-['Hlalkyl-GPC in intact macrophages, suggesting that the CoAindependent transacylation' system plays a very important part in the acylation of l-['H]alkyl-GPC in cells.We also confirmed that '"C-labeled 20:4(n -6), 20:5(n -3), 22:4(n -6), and 22:6(n -3) were transferred well from diacyl-GPC to l-alkyl-GPC in a CoAindependent manner.The transfer rates for 16:0,18:0, and 18:l from diacyl-GPC to l-alkyl-GPC were very low in the presence and absence of CoA.On the other hand, the transfer of 20:4 from diacyl-GPE or diacyl-GPI to l-alkyl-GPC or 1-acyl-GPC was markedly increased by the addition of CoA.The above results indicate that the transacylation system exhibits distinct donor and acceptor selectivities and CoA dependency.These transacylation reactions could be very important in the regulation of the levels and the availability of lysophospholipids, including lyso platelet-activating factor, and Czo and Czz polyunsaturated fatty acids in living cells.It has recently been demonstrated that several types of inflammatory and immunological cells including macrophages contain high amounts of ether phospholipids such as 1-0alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine (alkenylacyl-GPE)' and l-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC) (1).It seems, indeed, a unique feature of these cells that they contain significant amounts of alkylacyl-GPC, which is a minor component in various other tissues.
The production of phospholipid hydroperoxide and aldehydic phospholipid was examined in human red blood cell (RBC) membranes after peroxidation with 2, 2-azobis(2-amidino-propane)dihydrochloride (AAPH) or xanthine/xanthine oxidase (XO/XOD/Fe3+). Both radical-generation systems caused a profound decrease in the amount of polyunsaturated fatty acid (PUFA) in choline glycerophospholipid (CGP) and induced formation of peroxid-ized CGP in RBC membranes to different extents. No consistent generation of peroxidized lipids from CGP was evident after peroxidation with XO/XOD/Fe3+, which caused the apparent decomposition of phospholipids and the formation of large amounts of thiobar-bituric acid-reactive substance (TEARS). On the other hand, CGP hydroperoxide was formed as a primary product of peroxidation with AAPH. Aldehydic CGP was also detected as a secondary product of hydroperoxide decomposition in AAPH-peroxidized RBC membranes. Aldehydic CGP was preferentially generated from arachidonoyl CGP rather than from linoleoyl CGP in AAPH-peroxidized membranes. AAPH mainly oxidized CGP to hydroperoxide and aldehydic phospholipids. The sum of hydroperoxide and aldehyde of CGP corresponded to the loss of CGP due to peroxidation by AAPH. This result indicates that CGP was mainly converted into these two oxidized phospholipids in AAPH-peroxidized RBC membranes.
