Original Article
Ann Clin Biochem 1992; 29: 184-189
Role of reactive oxygen species in the generation of fluorescence by glycation C A Le Guen, A F Jones', A H Barnett- and J Lunec Wolfson Research Laboratories, Queen Elizabeth Medical Centre, Birmingham BJ5 2TH, 'Department of Clinical Chemistry, and 2Department of Medicine, East Birmingham Hospital and University of Birmingham, 45 Bordesley Green East, Birmingham B9 5ST, UK
Fluorescence (excitation 360 run, emission 454 nm) generation in glycated albumin was investigated. Antioxidants and the metal chelator desferrioxamine (DFX) were used to study the mechanism of fluorescence generation. Delipidation studies, reverse phase chromatography and scanning fluorimetry were performed to examine the nature of this fluorescence. The mechanism of action of aminoguanidine, a compound which has been shown to inhibit the formation of visible fluorescence in proteins in vitro and in vivo was investigated in relation to glycation and by comparison with compounds with structural similarities. We conclude that hydrogen peroxide, metal ions and hydroxyl radicals are involved in fluorescence generation in glycated albumin, which is largely lipid in nature, and arises through glycation, amino acid oxidation and changes in bound lipid. Our results suggest that the action of aminoguanidine is not specifically related to blocking of ketoamine groups on glycated proteins as previously suggested.
SUMMARY.
Additional key phrases: glycated albumin; antioxidants; delipidation; aminoguanidine
Increased fluorescence of skin (excitation 370 nm, emission 440 nm)' and serum proteins (excitation 360 nm, emission 454 nm)2 and increased lipid peroxidation products':" have been reported in diabetes mellitus. In skin collagen this fluorescence has been attributed to the Maillard reaction, whereas it was suggested that serum protein fluorescence is due to oxidative changes. Collagen and IgG fluorescence and lipid peroxides correlate with the presence of diabetic microangiopathy. Protein fluorescence can be generated by glycation and browning (Maillard reaction), by reaction with lipid peroxidation products' and by free radical mediated oxidation of amino acid residues." Browning can be inhibited by aminoguanidine, the proposed mechanism involving blocking of ketoamine carbonyl groups on glycated proteins." There is evidence that protein ketoamines can generate superoxide anion.v? Hydrogen peroxide and hydroxyl radicals can be generated from superoxide in the presence of catalytic metal ions. Metal ions and hydroxyl radicals are involved Correspondence: Dr C A Le Guen.
in fluorescence generation by glycation in albumin. 10, II As reactive oxygen species are potentially involved in fluorescence development.o" we have investigated the nature of this fluorescence, utilizing inhibitors of reactive oxygen species and have attempted to assess the role of lipid peroxidation and amino acid oxidation in this process in aged, glycated albumin.
MATERIALS Bovine serum albumin, essentially globulin free and essentially fat free, catalase, superoxide dismutase (SOD), guanidine hydrochloride, aminoguanidine hemisulphate, mannitol, and kynurenine were purchased from Sigma (Poole, Dorset, UK). Desferrioxamine was purchased from Ciba (Horsham, UK). Penicillin and streptomycin were purchased from Evans (Speke, Merseyside, UK). All other reagents were purchased from BDH (Poole, Dorset, UK). HPLC equipment was purchased from Anachem (Luton, Beds, UK) and Technicol (Stockport, Cheshire, UK).
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Reactive oxygen species and protein browning
METHODS Preparation of glycated protein Albumin (40 g/L) was incubated with 500 mmol/L glucose, in phosphate buffered saline pH 7· 4, for 3 days at 37°C. A high concentration of glucose was used to achieve levels of glycation comparable with long lived proteins, which are exposed to prolonged hyperglycaernia. This was then dialysed against a large volume of phosphate buffered saline for 24 h, with mixing, to remove excess glucose. This process yielded a protein with a fructosamine value of 10'4. Non-glycated protein for comparison was treated similarly, without the addition of glucose. Penicillin and streptomycin were added during preparation and subsequent incubation to inhibit bacterial growth. Ageing of proteins Protein was aged for 10 days at 37 c C . Ten days was chosen as an optimum incubation time, as catalase activity was negligibleat longer incubation times. Penicillin and streptomycin were again added to inhibit bacterial growth. Protein was incubated alone or with one of the inhibitors under study. To investigate the involvement of reactive oxygen species, glycated albumin was incubated alone or with 100 mg/L superoxide dismutase (to inhibit superoxide anion), 500 mg/L catalase (to decompose hydrogen peroxide), 50 mmoIlL thiourea, 50 mmoIlL mannitol (hydroxyl radical scavengers) or 50 ILmoIlL desferrioxamine (metal ion chelator, putative inhibitor of hydroxyl radical generation via the Fenton reaction). 12 To investigate the effect of denaturation, glycated and non-glycated albumin were incubated alone or with 200 mmoIlL aminoguanidine, 200 mmoIlL guanidine or 200 mmoIlL urea. The concentration of aminoguanidine was based on that used in a previous study, 7 denaturing agents were used at similar concentrations for comparison. This concentration is lower than that commonly used for denaturation or delipidation (one fifth of that used in Reference 8), but in view of the longer time course, we thought that delipidation by guanidine (or aminoguanidine, which has a similar structure), might be significant. The role of bound lipid was investigated using fat free albumin and native albumin. Specimens were analysed as previously described for serum proteins,'! fluorescence (excitation 325-395 nm, emission 430-470 nm) and uv absorbance (280 nm) being measured. Results in the first two sections are expressed as the increase in
185
the ratio of visible fluorescence intensity to uv absorbance upon incubation, in arbitrary units ± standard deviation, as albumin has significant fluorescence associated with it prior to incubation. The ratio of fluorescence to uv absorbance corrects for differences in protein concentration. No increase in absorbance at 280 nm during the course of the experiment, due to browning was observed, so this correction was thought to be valid. Intra-batch and inter-batch coefficients of variation for this method have been calculated by experiment to be 3070 and 5070, respectively. Results were analysed using Student's t test. In addition excitation and emission scans were performed on albumin which had been incubated ± 20 mmoIlL glucose for 2 or 5 weeks. Excitation scans were performed between 290 and 400 nm with emission at 440 nm. Emission scans were performed between 400 and 600 nm with excitation at 360 nm. To investigate whether the fluorescence was due to Schiff bases, e.g, aminoimino-propene adducts derived from lipid hydroperoxides, 10 ILL of 1 moIlL sodium hydroxide was added and the scan repeated, followed by neutralization with 10 ILL of I moIlL hydrochloric acid and a repeat scan.!" This was performed on each protein after 5 weeks' incubation. Identification of kynurenine Reverse phase chromatography was carried out on albumin digests, using an increasing concentration of acetonitrile in phosphate buffer, coupled to detection by absorbance at 240 nm and filter fluorimetry (excitation 325-395 nm, emission 430-470 nm)." Kynurenine was identified in pronase digests by 'spiking' with authentic standard to give a concentration of added kynurenine of 10 mg/L (digestion was as used for albumin)."
RESULTS Involvement and identification of reactive oxygen species in fluorescence generation The effect of a range of antioxidant enzymes, free radical scavengers and the metal chelator desferrioxamine on fluorescence generation in aged glycated albumin is shown in Fig. 1. Addition of superoxide dismutase to protein undergoing incubation was associated with an 85070 greater increase in fluorescence ratio compared with control (P
Ann Clin Biochem 1992; 29: 184-189
Role of reactive oxygen species in the generation of fluorescence by glycation C A Le Guen, A F Jones', A H Barnett- and J Lunec Wolfson Research Laboratories, Queen Elizabeth Medical Centre, Birmingham BJ5 2TH, 'Department of Clinical Chemistry, and 2Department of Medicine, East Birmingham Hospital and University of Birmingham, 45 Bordesley Green East, Birmingham B9 5ST, UK
Fluorescence (excitation 360 run, emission 454 nm) generation in glycated albumin was investigated. Antioxidants and the metal chelator desferrioxamine (DFX) were used to study the mechanism of fluorescence generation. Delipidation studies, reverse phase chromatography and scanning fluorimetry were performed to examine the nature of this fluorescence. The mechanism of action of aminoguanidine, a compound which has been shown to inhibit the formation of visible fluorescence in proteins in vitro and in vivo was investigated in relation to glycation and by comparison with compounds with structural similarities. We conclude that hydrogen peroxide, metal ions and hydroxyl radicals are involved in fluorescence generation in glycated albumin, which is largely lipid in nature, and arises through glycation, amino acid oxidation and changes in bound lipid. Our results suggest that the action of aminoguanidine is not specifically related to blocking of ketoamine groups on glycated proteins as previously suggested.
SUMMARY.
Additional key phrases: glycated albumin; antioxidants; delipidation; aminoguanidine
Increased fluorescence of skin (excitation 370 nm, emission 440 nm)' and serum proteins (excitation 360 nm, emission 454 nm)2 and increased lipid peroxidation products':" have been reported in diabetes mellitus. In skin collagen this fluorescence has been attributed to the Maillard reaction, whereas it was suggested that serum protein fluorescence is due to oxidative changes. Collagen and IgG fluorescence and lipid peroxides correlate with the presence of diabetic microangiopathy. Protein fluorescence can be generated by glycation and browning (Maillard reaction), by reaction with lipid peroxidation products' and by free radical mediated oxidation of amino acid residues." Browning can be inhibited by aminoguanidine, the proposed mechanism involving blocking of ketoamine carbonyl groups on glycated proteins." There is evidence that protein ketoamines can generate superoxide anion.v? Hydrogen peroxide and hydroxyl radicals can be generated from superoxide in the presence of catalytic metal ions. Metal ions and hydroxyl radicals are involved Correspondence: Dr C A Le Guen.
in fluorescence generation by glycation in albumin. 10, II As reactive oxygen species are potentially involved in fluorescence development.o" we have investigated the nature of this fluorescence, utilizing inhibitors of reactive oxygen species and have attempted to assess the role of lipid peroxidation and amino acid oxidation in this process in aged, glycated albumin.
MATERIALS Bovine serum albumin, essentially globulin free and essentially fat free, catalase, superoxide dismutase (SOD), guanidine hydrochloride, aminoguanidine hemisulphate, mannitol, and kynurenine were purchased from Sigma (Poole, Dorset, UK). Desferrioxamine was purchased from Ciba (Horsham, UK). Penicillin and streptomycin were purchased from Evans (Speke, Merseyside, UK). All other reagents were purchased from BDH (Poole, Dorset, UK). HPLC equipment was purchased from Anachem (Luton, Beds, UK) and Technicol (Stockport, Cheshire, UK).
184
Downloaded from acb.sagepub.com by guest on June 5, 2016
Reactive oxygen species and protein browning
METHODS Preparation of glycated protein Albumin (40 g/L) was incubated with 500 mmol/L glucose, in phosphate buffered saline pH 7· 4, for 3 days at 37°C. A high concentration of glucose was used to achieve levels of glycation comparable with long lived proteins, which are exposed to prolonged hyperglycaernia. This was then dialysed against a large volume of phosphate buffered saline for 24 h, with mixing, to remove excess glucose. This process yielded a protein with a fructosamine value of 10'4. Non-glycated protein for comparison was treated similarly, without the addition of glucose. Penicillin and streptomycin were added during preparation and subsequent incubation to inhibit bacterial growth. Ageing of proteins Protein was aged for 10 days at 37 c C . Ten days was chosen as an optimum incubation time, as catalase activity was negligibleat longer incubation times. Penicillin and streptomycin were again added to inhibit bacterial growth. Protein was incubated alone or with one of the inhibitors under study. To investigate the involvement of reactive oxygen species, glycated albumin was incubated alone or with 100 mg/L superoxide dismutase (to inhibit superoxide anion), 500 mg/L catalase (to decompose hydrogen peroxide), 50 mmoIlL thiourea, 50 mmoIlL mannitol (hydroxyl radical scavengers) or 50 ILmoIlL desferrioxamine (metal ion chelator, putative inhibitor of hydroxyl radical generation via the Fenton reaction). 12 To investigate the effect of denaturation, glycated and non-glycated albumin were incubated alone or with 200 mmoIlL aminoguanidine, 200 mmoIlL guanidine or 200 mmoIlL urea. The concentration of aminoguanidine was based on that used in a previous study, 7 denaturing agents were used at similar concentrations for comparison. This concentration is lower than that commonly used for denaturation or delipidation (one fifth of that used in Reference 8), but in view of the longer time course, we thought that delipidation by guanidine (or aminoguanidine, which has a similar structure), might be significant. The role of bound lipid was investigated using fat free albumin and native albumin. Specimens were analysed as previously described for serum proteins,'! fluorescence (excitation 325-395 nm, emission 430-470 nm) and uv absorbance (280 nm) being measured. Results in the first two sections are expressed as the increase in
185
the ratio of visible fluorescence intensity to uv absorbance upon incubation, in arbitrary units ± standard deviation, as albumin has significant fluorescence associated with it prior to incubation. The ratio of fluorescence to uv absorbance corrects for differences in protein concentration. No increase in absorbance at 280 nm during the course of the experiment, due to browning was observed, so this correction was thought to be valid. Intra-batch and inter-batch coefficients of variation for this method have been calculated by experiment to be 3070 and 5070, respectively. Results were analysed using Student's t test. In addition excitation and emission scans were performed on albumin which had been incubated ± 20 mmoIlL glucose for 2 or 5 weeks. Excitation scans were performed between 290 and 400 nm with emission at 440 nm. Emission scans were performed between 400 and 600 nm with excitation at 360 nm. To investigate whether the fluorescence was due to Schiff bases, e.g, aminoimino-propene adducts derived from lipid hydroperoxides, 10 ILL of 1 moIlL sodium hydroxide was added and the scan repeated, followed by neutralization with 10 ILL of I moIlL hydrochloric acid and a repeat scan.!" This was performed on each protein after 5 weeks' incubation. Identification of kynurenine Reverse phase chromatography was carried out on albumin digests, using an increasing concentration of acetonitrile in phosphate buffer, coupled to detection by absorbance at 240 nm and filter fluorimetry (excitation 325-395 nm, emission 430-470 nm)." Kynurenine was identified in pronase digests by 'spiking' with authentic standard to give a concentration of added kynurenine of 10 mg/L (digestion was as used for albumin)."
RESULTS Involvement and identification of reactive oxygen species in fluorescence generation The effect of a range of antioxidant enzymes, free radical scavengers and the metal chelator desferrioxamine on fluorescence generation in aged glycated albumin is shown in Fig. 1. Addition of superoxide dismutase to protein undergoing incubation was associated with an 85070 greater increase in fluorescence ratio compared with control (P
