ANALYTICAL
192,
BIOCHEMISTRY
109-111
(1991)
A Microassay for Protein Glycation on the Periodate Method Nessar
Ahmed
Oxford Research
Received
June
and Anna Unit,
J. Furth
The Open University,
Boars Hill,
Oxford OX1 5HR, England
18, 1990
The periodate assay for glycated protein has been adapted for use with a microplate reader. Up to 94 samples can be read in 40 s, and sensitivity has been improved so that only 2-40 nmol of protein-bound sugar are needed per well. Yields have been increased to over 90%, allowing in vitro glycation of human serum albuo ISSI Academic min to be assayed on O.l-mg aliquots. Press,
Inc.
Protein glycation is the nonenzymatic addition of single sugar residues to protein amino groups and occurs spontaneously wherever protein molecules are exposed to high concentrations of reducing sugar for prolonged periods, e.g., in diabetes and aging (1). However, similar glycation-promoting conditions are found in the glucose-rich culture media used for cell cloning, during long-term storage of certain plant products, and during the intracellular buildup of glycolytic intermediates (2). Although glycation could alter protein structure and function in any of these situations, it is very difficult to detect, particularly in the presence of glycoproteins. All the reliable glycation assays (reviewed in (3)) need milligram quantities of protein. Exceptions may make use of HPLC equipment, but even then the protein must first be hydrolyzed. Of the simple calorimetric assays for protein-bound sugar, the periodate assay devised by Gallop et ~2.in 1981 has many advantages (4). It quantifies the formaldehyde released by periodate oxidation of C-l hydroxyls in the Amadori product form of glycated proteins. The HCHO is converted to a chromophore (DDL)’ by reaction with acetylacetone in ammonia. Schiff base forms of glycated protein will interfere and should be removed by dialysis against distilled water (5) or buffer at pH 5 (6). This pretreatment dissociates the ’ Abbreviations dihydrolutidine.
Based
used:
BSA,
bovine
0003-2697191 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
serum
albumin;
DDL,
diacetyl-
Schiff base, and also removes small-molecule contaminants that may interfere in the assay. Glycation assays on undialyzed samples (as in the serum fructosamine assay) are frequently subject to interference by smallmolecule contaminants (e.g., (7)). Glycoproteins are also a potential problem, but will not interfere in the periodate assay unless they carry chain-terminal reducing sugars or sialic acid derivatives. In the original Gallop procedure, DDL is detected fluorometrically. Even then, some 20 nmol of protein are needed per assay, and a fluorometer must be available. We have adapted the periodate assay for use with a microplate reader, making use of the high molar absorbance of DDL at 412 nm (8). Only nanomolar quantities (0.1 mg of albumin) are needed per assay, and 47 duplicates can be read in a few seconds.
MATERIALS
AND
METHODS
Chemicals and equipment. Acetylacetone and bovine serum albumin (fraction V) were obtained from BDH (Poole, UK). Human serum albumin was a gift from the National Institute for Biological Standards and Control (Potters Bar, UK). Absorbance was read in a Biotek automated microplate reader Model EL 311, using a 405-nm filter, or on a Unicam SP 300 spectrophotometer. Eppendorf tubes were centrifuged in a Heraeus Biofuge A bench-top centrifuge. In vitro gzycation. Standard preparations of a defined glycated protein, bovine serum albumin, were made by incubating BSA (10 mg/ml) in 0.5 M glucose for 15 days at 37°C in 0.05 M phosphate buffer, pH 7.4 containing 3 IIIM sodium azide. Samples were dialyzed extensively against water to remove Schiff base and free glucose, and stored frozen. Molar absorbance of DDL at 405 nm. This was calculated from the published molar absorbance at 412 nm (8), by using a spectrophotometer to compare the absorbance of DDL at 412 nm (the X,,,) and 405 nm (the 109
Inc. reserved.
110
AHMED
AND
0.6
E g 2
0.4
0.2
10
20 nmols
FIG.
1.
nanomoles microplate
30 /
40
well
Standard curve showing absorbance of fructose (0) or formaldehyde (0), reader.
at 405 nm against as read on the Biotek
wavelength of the nearest Biotek filter). This method gave the molar absorbance of DDL at 405 nm as 7780. Protein assays. Protein concentration was measured by the bicinchoninic acid method using the Pierce BCA protein assay reagent (9). Glycation assay procedure. The volume of sample aliquots containing 2-40 nmol of fructose or Amadori product was adjusted to 40 ~1 with water and incubated with 20 ~1 of 0.1 M HCl and 20 ~1 of 0.05 M NaIO, at room temperature for 30 min. To terminate oxidation, samples were cooled in ice for 10 min, and mixed with 20 ~1 of 15% ZnSO, and 20 ~1 of 0.7 M NaOH. Both reagents were precooled, and samples were vortexed throughout the addition. They were centrifuged for 10 min at 9OOOg to remove precipitated zinc periodate, and loo-p1 aliquots of supernatant fluid were transferred to microplate wells. Formaldehyde detection reagent was freshly prepared by mixing 46 ~1of acetylacetone in 10 ml of 3.3 M ammonium acetate; 200 ~1 of this reagent was added to each well and mixed thoroughly with the sample, and DDL allowed to develop by incubating for 1 h at 37°C. Reagent blanks (100 ~1 water and 200 ~1 detection reagent) were incubated at the same time. The microplate was inserted into the reader, and absorbance of reagent blanks at 405 nm subtracted automatically from absorbance of sample solutions. RESULTS
AND
FURTH
ates 1 mol of HCHO per mole on periodate oxidation, and, therefore, can be used as a standard for the Amadori product. Figure 1 shows that with the microassay procedure, absorbance is linear for 5-40 nmol of fructose per well (i.e., 0.1-l nmol/pl per 40-~1 aliquot). As little as 2 nmol can be readily detected (giving absorbance readings of 0.04); at lower concentrations sensitivity is constrained partly by the short light path (0.88 cm for 300 ~1 of solution per well) and partly by the yield of HCHO from fructose. Theoretically, the yield may be reduced at any of three stages, i.e., by incomplete oxidation and cleavage of C-l to produce HCHO, by adsorption or trapping of free HCHO in the zinc periodate precipitate, or by incomplete conversion of HCHO to DDL. We measured the yield in this last step by incubating standard HCHO solutions with detection reagent. The absorbance readings differed little from those calculated using the molar absorbance of DDL, and assuming 100% conversion of HCHO to DDL. However, when HCHO was produced indirectly from fructose, absorbance values were some 5% lower (Figure l), showing that material is lost during oxidation and/or centrifugation. Loss of HCHO on the zinc periodate precipitate can be minimized by high-speed centrifugation to reduce precipitate bulk. A greater proportion of HCHO-containing solution can thus be removed as supernatant liquid and converted to DDL. This modification allowed us to improve our overall yield (from fructose to DDL) from 67% with the Gallop procedure, to 90% with the microassay procedure. This compares favorably with yields in other methods, e.g., 30% for the furosine assay
0.40-
0.32.
0.24E g 4
0.16.
DISCUSSION 80
Fructose resembles the Amadori product form of a glycated protein in having a C-2 carbonyl group in the straight-chain configuration, but existing predominantly in ring form. Like the Amadori product, it liber-
Protein
FIG. 2.
160 Assayed
240
320
(ug)
Effect of increasing protein in assay. Differing amounts of in vitro glycated BSA were assayed in the same 40-pl volume, under the standard procedure.
MICROASSAY
FOR
(lo), and 80% for the thiobarbituric method under optimum conditions (11). Using only 0.1 mg of protein, we estimated the in uiuo glycation of human serum albumin as 0.39 mol per mole. This is in the upper range of published values (summarized in (12) and estimated by a variety of methods). Our intraassay coefficient of variation was 1.1% (n = 5). In the range O-O.3 mg of protein per well, absorbance was linear with concentrations of glycated protein (Fig. 2). The microassay can therefore be used to quantify glycation on small amounts of intact protein, without the need for prior hydrolysis or expensive equipment such as HPLC. With a microplate reader, up to 94 samples can be read simultaneously.
M., Cerami, tab. Rev. 4,437T451.
111
GLYCATION
2. McDonald, M., Shapiro, R., Bleichmann, M., Bunn, H. F. (1978) J. Biol. Chem. 253,2327-2332. 3. Furth,
A. J. (1988)
Anal.
Biochem.
Solway,
J., and
175, 1988, 347-360.
4. Gallop, P. M., Fluckiger, R., Hanneken, A., Mininsohn, M. M., and Gabbay, K. H. (1981) Anal. Biochem. 117,427-432. 5. McPherson, J. D., Shilton, B. H., and Walton, D. J. (1988) Biochemistry 27, 1901-1907. 6. Bisse, E., Berger, W., and Fluckiger, R. (1982) Diabetes 31,630633. 7. Brighton, M. W., and Furth, A. J. (1988) Clin. Chem. 34, 2378. 8. Nash, T. (1953) Biochem. J. 55,416-421. 9. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) AnaL Biochem. 150,76-85. 10. Schleicher, E., and Wieland, 0. H. (1981) J. Clin. Chem. Clin. Biochem. 19,81-87. 11. Ney, K. A., Colley, K. J., and Pizza, S. V. (1981) Anal. Biochem.
118,294-300.
REFERENCES 1. Brownlee,
PROTEIN
C., and Vlassara,
H. (1988)
DiabeteslMe-
12. Baynes, J. W., et al. (1989) in The Maillard Reaction in Ageing, Diabetes and Nutrition (Baynes, J. W., and Monnier, V. M., Eds.), pp. 43-67, A. R. Liss, New York.
192,
BIOCHEMISTRY
109-111
(1991)
A Microassay for Protein Glycation on the Periodate Method Nessar
Ahmed
Oxford Research
Received
June
and Anna Unit,
J. Furth
The Open University,
Boars Hill,
Oxford OX1 5HR, England
18, 1990
The periodate assay for glycated protein has been adapted for use with a microplate reader. Up to 94 samples can be read in 40 s, and sensitivity has been improved so that only 2-40 nmol of protein-bound sugar are needed per well. Yields have been increased to over 90%, allowing in vitro glycation of human serum albuo ISSI Academic min to be assayed on O.l-mg aliquots. Press,
Inc.
Protein glycation is the nonenzymatic addition of single sugar residues to protein amino groups and occurs spontaneously wherever protein molecules are exposed to high concentrations of reducing sugar for prolonged periods, e.g., in diabetes and aging (1). However, similar glycation-promoting conditions are found in the glucose-rich culture media used for cell cloning, during long-term storage of certain plant products, and during the intracellular buildup of glycolytic intermediates (2). Although glycation could alter protein structure and function in any of these situations, it is very difficult to detect, particularly in the presence of glycoproteins. All the reliable glycation assays (reviewed in (3)) need milligram quantities of protein. Exceptions may make use of HPLC equipment, but even then the protein must first be hydrolyzed. Of the simple calorimetric assays for protein-bound sugar, the periodate assay devised by Gallop et ~2.in 1981 has many advantages (4). It quantifies the formaldehyde released by periodate oxidation of C-l hydroxyls in the Amadori product form of glycated proteins. The HCHO is converted to a chromophore (DDL)’ by reaction with acetylacetone in ammonia. Schiff base forms of glycated protein will interfere and should be removed by dialysis against distilled water (5) or buffer at pH 5 (6). This pretreatment dissociates the ’ Abbreviations dihydrolutidine.
Based
used:
BSA,
bovine
0003-2697191 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
serum
albumin;
DDL,
diacetyl-
Schiff base, and also removes small-molecule contaminants that may interfere in the assay. Glycation assays on undialyzed samples (as in the serum fructosamine assay) are frequently subject to interference by smallmolecule contaminants (e.g., (7)). Glycoproteins are also a potential problem, but will not interfere in the periodate assay unless they carry chain-terminal reducing sugars or sialic acid derivatives. In the original Gallop procedure, DDL is detected fluorometrically. Even then, some 20 nmol of protein are needed per assay, and a fluorometer must be available. We have adapted the periodate assay for use with a microplate reader, making use of the high molar absorbance of DDL at 412 nm (8). Only nanomolar quantities (0.1 mg of albumin) are needed per assay, and 47 duplicates can be read in a few seconds.
MATERIALS
AND
METHODS
Chemicals and equipment. Acetylacetone and bovine serum albumin (fraction V) were obtained from BDH (Poole, UK). Human serum albumin was a gift from the National Institute for Biological Standards and Control (Potters Bar, UK). Absorbance was read in a Biotek automated microplate reader Model EL 311, using a 405-nm filter, or on a Unicam SP 300 spectrophotometer. Eppendorf tubes were centrifuged in a Heraeus Biofuge A bench-top centrifuge. In vitro gzycation. Standard preparations of a defined glycated protein, bovine serum albumin, were made by incubating BSA (10 mg/ml) in 0.5 M glucose for 15 days at 37°C in 0.05 M phosphate buffer, pH 7.4 containing 3 IIIM sodium azide. Samples were dialyzed extensively against water to remove Schiff base and free glucose, and stored frozen. Molar absorbance of DDL at 405 nm. This was calculated from the published molar absorbance at 412 nm (8), by using a spectrophotometer to compare the absorbance of DDL at 412 nm (the X,,,) and 405 nm (the 109
Inc. reserved.
110
AHMED
AND
0.6
E g 2
0.4
0.2
10
20 nmols
FIG.
1.
nanomoles microplate
30 /
40
well
Standard curve showing absorbance of fructose (0) or formaldehyde (0), reader.
at 405 nm against as read on the Biotek
wavelength of the nearest Biotek filter). This method gave the molar absorbance of DDL at 405 nm as 7780. Protein assays. Protein concentration was measured by the bicinchoninic acid method using the Pierce BCA protein assay reagent (9). Glycation assay procedure. The volume of sample aliquots containing 2-40 nmol of fructose or Amadori product was adjusted to 40 ~1 with water and incubated with 20 ~1 of 0.1 M HCl and 20 ~1 of 0.05 M NaIO, at room temperature for 30 min. To terminate oxidation, samples were cooled in ice for 10 min, and mixed with 20 ~1 of 15% ZnSO, and 20 ~1 of 0.7 M NaOH. Both reagents were precooled, and samples were vortexed throughout the addition. They were centrifuged for 10 min at 9OOOg to remove precipitated zinc periodate, and loo-p1 aliquots of supernatant fluid were transferred to microplate wells. Formaldehyde detection reagent was freshly prepared by mixing 46 ~1of acetylacetone in 10 ml of 3.3 M ammonium acetate; 200 ~1 of this reagent was added to each well and mixed thoroughly with the sample, and DDL allowed to develop by incubating for 1 h at 37°C. Reagent blanks (100 ~1 water and 200 ~1 detection reagent) were incubated at the same time. The microplate was inserted into the reader, and absorbance of reagent blanks at 405 nm subtracted automatically from absorbance of sample solutions. RESULTS
AND
FURTH
ates 1 mol of HCHO per mole on periodate oxidation, and, therefore, can be used as a standard for the Amadori product. Figure 1 shows that with the microassay procedure, absorbance is linear for 5-40 nmol of fructose per well (i.e., 0.1-l nmol/pl per 40-~1 aliquot). As little as 2 nmol can be readily detected (giving absorbance readings of 0.04); at lower concentrations sensitivity is constrained partly by the short light path (0.88 cm for 300 ~1 of solution per well) and partly by the yield of HCHO from fructose. Theoretically, the yield may be reduced at any of three stages, i.e., by incomplete oxidation and cleavage of C-l to produce HCHO, by adsorption or trapping of free HCHO in the zinc periodate precipitate, or by incomplete conversion of HCHO to DDL. We measured the yield in this last step by incubating standard HCHO solutions with detection reagent. The absorbance readings differed little from those calculated using the molar absorbance of DDL, and assuming 100% conversion of HCHO to DDL. However, when HCHO was produced indirectly from fructose, absorbance values were some 5% lower (Figure l), showing that material is lost during oxidation and/or centrifugation. Loss of HCHO on the zinc periodate precipitate can be minimized by high-speed centrifugation to reduce precipitate bulk. A greater proportion of HCHO-containing solution can thus be removed as supernatant liquid and converted to DDL. This modification allowed us to improve our overall yield (from fructose to DDL) from 67% with the Gallop procedure, to 90% with the microassay procedure. This compares favorably with yields in other methods, e.g., 30% for the furosine assay
0.40-
0.32.
0.24E g 4
0.16.
DISCUSSION 80
Fructose resembles the Amadori product form of a glycated protein in having a C-2 carbonyl group in the straight-chain configuration, but existing predominantly in ring form. Like the Amadori product, it liber-
Protein
FIG. 2.
160 Assayed
240
320
(ug)
Effect of increasing protein in assay. Differing amounts of in vitro glycated BSA were assayed in the same 40-pl volume, under the standard procedure.
MICROASSAY
FOR
(lo), and 80% for the thiobarbituric method under optimum conditions (11). Using only 0.1 mg of protein, we estimated the in uiuo glycation of human serum albumin as 0.39 mol per mole. This is in the upper range of published values (summarized in (12) and estimated by a variety of methods). Our intraassay coefficient of variation was 1.1% (n = 5). In the range O-O.3 mg of protein per well, absorbance was linear with concentrations of glycated protein (Fig. 2). The microassay can therefore be used to quantify glycation on small amounts of intact protein, without the need for prior hydrolysis or expensive equipment such as HPLC. With a microplate reader, up to 94 samples can be read simultaneously.
M., Cerami, tab. Rev. 4,437T451.
111
GLYCATION
2. McDonald, M., Shapiro, R., Bleichmann, M., Bunn, H. F. (1978) J. Biol. Chem. 253,2327-2332. 3. Furth,
A. J. (1988)
Anal.
Biochem.
Solway,
J., and
175, 1988, 347-360.
4. Gallop, P. M., Fluckiger, R., Hanneken, A., Mininsohn, M. M., and Gabbay, K. H. (1981) Anal. Biochem. 117,427-432. 5. McPherson, J. D., Shilton, B. H., and Walton, D. J. (1988) Biochemistry 27, 1901-1907. 6. Bisse, E., Berger, W., and Fluckiger, R. (1982) Diabetes 31,630633. 7. Brighton, M. W., and Furth, A. J. (1988) Clin. Chem. 34, 2378. 8. Nash, T. (1953) Biochem. J. 55,416-421. 9. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) AnaL Biochem. 150,76-85. 10. Schleicher, E., and Wieland, 0. H. (1981) J. Clin. Chem. Clin. Biochem. 19,81-87. 11. Ney, K. A., Colley, K. J., and Pizza, S. V. (1981) Anal. Biochem.
118,294-300.
REFERENCES 1. Brownlee,
PROTEIN
C., and Vlassara,
H. (1988)
DiabeteslMe-
12. Baynes, J. W., et al. (1989) in The Maillard Reaction in Ageing, Diabetes and Nutrition (Baynes, J. W., and Monnier, V. M., Eds.), pp. 43-67, A. R. Liss, New York.
