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An enzymatic method for the measurement of glycated albumin in biological samples
Clinica Chimica Acta 324 (2002) 61 – 71 www.elsevier.com/locate/clinchim
An enzymatic method for the measurement of glycated albumin in biological samples Takuji Kouzuma*, Tomomi Usami, Masaru Yamakoshi, Mamoru Takahashi, Shigeyuki Imamura Diagnostics R&D Department, Fine Chemicals and Diagnostics Division, Health Care Company, Asahi Kasei Corporation. 632-1, Mifuku, Ohito-cho, Tagata-gun, Shizuoka-ken, 410-2321, Japan Received 8 April 2002; received in revised form 10 June 2002; accepted 10 June 2002
Abstract Background: In order to determine glycated albumin more easily and rapidly, we developed a new enzymatic method for glycated albumin in blood samples. Methods: The method involves use of albumin-specific proteinase, ketoamine oxidase and serum albumin assay reagent. In the assay, glycated albumin is hydrolyzed to glycated amino acids by proteinase digestion, and ketoamine oxidase oxidizes the glycated amino acids to produce hydrogen peroxide, which is quantitatively measured. Glycated albumin is calculated as the percentage of glycated albumin in total albumin. Results: The calibration curve for glycated albumin concentration was linear (rp=0.999) between 0.0 and 50.0 g/l and that for albumin concentration was linear (rp=0.999) between 0.0 and 60.0 g/l. The analytical recoveries of exogenous glycated albumin added to serum were 100 – 102.5%. The within-run and between-run CVs were 0.45 – 0.67% and 1.09 – 1.26%, respectively. This method was free from interference by bilirubin, chyle, glucose, globulins and labile intermediate. Weak interference by hemoglobin and ascorbic acid was observed. Glycated albumin detected by the present method was significantly correlated with glycated albumin detected by highperformance liquid-chromatographic (HPLC) method (serum: rs=0.989, plasma: rp=0.992). Conclusions: This new enzymatic method is simple, rapid, allows multiple determinations and enables quantitative analysis of glycated albumin. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Glycated albumin; Proteinase; Ketoamine oxidase
1. Introduction Chronic hyperglycemia in diabetes results in increased concentrations of nonenzymatically glycated proteins including hemoglobin [1] and albumin [2,3]. Since the modification of hemoglobin by glucose occurs continually during the life span of * Corresponding author. Tel.: +81-0558-76-8607; fax: +810558-76-7149. E-mail address: [email protected] (T. Kouzuma).
the erythrocyte, glycated hemoglobin concentrations provide a time-averaged index of the degree of hyperglycemia during the previous 2 months in humans [4]. In the same way, glycated albumin appears to provide an index of the state of glycemic control for approximately the previous 2 weeks. The concentration of glycated albumin should provide additional useful information on glycemic control when monitoring effects of changes in diet or insulin therapy. The serum concentration of fructosamine may also be used as an index of glycemic control for the
0009-8981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 8 9 8 1 ( 0 2 ) 0 0 2 0 7 - 3
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previous 2 weeks, but fructosamine concentrations are strongly influenced by the concentrations of protein, bilirubin, hemoglobin, uric acid and other low-molecular-weight substances coexisting in the blood and are, therefore, less accurate [5]. In order to avoid effects of low-molecular-weight substances coexisting in the blood, an enzymatic assay for fructosamine using proteinase and ketoamine oxidase [6,7] was developed. However, since fructosamine concentrations determined by the enzymatic method are strongly influenced by concentration of protein and the exact half-lives of all glycated proteins have not yet been determined, measurement of glycated albumin may be more useful clinically. The concentrations of glycated albumin in biological specimens have been measured by affinity chromatography [5,8], ion exchange chromatography [9,10], thiobarbituric acid assay [11,12], radioimmunoassay [13,14] and boronate immunoassay [15]. However, these methods have a number of disadvantages, e.g. specimens must be pretreated and procedures are complicated. A simple high-performance liquid-chromatographic (HPLC) method for separation of glycated albumin involving a combination of ion-exchange chromatography to separate albumin and boronate affinity chromatography to separate glycated albumin from nonglycated albumin [16,17] has been established. However, this method could measure only 12 samples/h, a rate not clearly sufficient for routine clinical use. An enzymatic method for glycated albumin has not been reported. In order to determine glycated albumin more easily and rapidly, we developed a new enzymatic method for glycated albumin using albumin-specific proteinase, ketoamine oxidase and albumin assay reagent. We describe the screening of albumin-specific proteinase and detergent, optimization studies and the assay evaluation of this enzymatic method.
2. Materials and methods 2.1. Materials Common reagents, albumin assay reagent and fructosamine assay reagent were from Wako (Osaka, Japan). Ascorbic acid oxidase (cucurbita species)
was from Roche Diagnostics (Mannheim, Germany). Folin and Ciocalteu’s phenol reagent, albumin (human; essentially globulin-free), globulins (human; Cohn Fraction II, III, IV and g-globulins), proteinase (protease type X X VUU) and peroxidase (Type II from horseradish) were from Sigma (St. Louis, MO). Interference Check-A Plus (free and conjugated bilirubin, hemoglobin and chyle) was from Kokusai Shiyaku, (Kobe, Japan). N,N-Bis(4-sulfobutyl)-3methylanilin, disodium salt (TODB) and 3-[(3-Cholamido-propyl) dimethyl-ammonio]-2-hydroxypropanesulfonic acid (CHAPSO), were from Dojindo Laboratories (Kumamoto, Japan). Ultrafree MC centrifugal filter unit was from Millipore (Bedford, MA). Fresh, frozen human plasma was from Veritas (Tokyo, Japan). Ketoamine oxidase was from Asahi Kasei (Tokyo, Japan). Blood samples were separated by centrifugation at 3000g at 4 jC. All samples were collected as spot samples and stored at 80 jC without antiseptics until assay. 2.2. Substrates q-N-(1-deoxy-D-fructosyl)-a-Z-lysine (FZL) was, essentially, prepared by the method of Hashiba [18]. Human serum globulins were prepared by the method of Shima et al. [16]. Globulins were separate from albumin by anion exchange column. Nonglycated albumin and glycated albumin were purified from fresh, frozen human plasma. First, human serum albumin was prepared by the method of Matsuda and Takahashi [19]. Then, 1.0 g of human serum albumin was dissolved in 100 ml of 0.05 mol/l glycine – NaOH containing 20 g/l MgCl2 (pH 8.5) and incubated with stirring for 0.5 h at 4 jC with 70 ml of phenylboronic acid resin equilibrated with the same buffer. The solution was passed through a glass filter and the filtrate was pooled as a nonglycated albumin solution. Phenylboronic acid resin was suspended in 30 ml of 0.02 mol/l Tris(hydroxymethyl)aminomethane (Tris) buffer containing 150 mmol/l sorbitol and 440 mmol/l NaCl (pH 8.5) and incubated with stirring for 0.5 h at 4 jC The solution was passed through a glass filter and the filtrate was pooled as a glycated albumin solution. The glycated albumin solution and nonglycated albumin solution were then repurified in the same manner. The final
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solution obtained was concentrated to 40 g/l by an ultrafiltration system. The purified nonglycated albumin solution and glycated albumin solution were assayed with a fructosamine assay kit and HPLC method (GA value of nonglycated albumin solution and glycated albumin solution were 4.3% and 95.4%, respectively). A high GA value control was prepared by incubation of fresh, frozen human plasma with 400 mmol/l glucose (100 mmol/l phosphate buffer pH 7.5) at 37 jC for 14 days. The reaction mixture was dialyzed against 20 mmol/l Tris – HCl buffer (pH 7.5) and lyophilized. 2.3. Definition of proteinase and ketoamine oxidase activity 2.3.1. Proteinase activity The proteinase activity was determined by caseinFolin method. One unit (1U) of proteinase was defined as the amount of enzyme that catalyzed casein to produce color equivalent to 1.0 Ag of tyrosine per minute at 30 jC (color by Folin and Ciocalteu’s phenol reagent). 2.3.2. Ketoamine oxidase activity Ketoamine oxidase activity was monitored by the release of H2O2. One unit (1U) of ketoamine oxidase was defined as the amount of enzyme that oxidizes 1 Amol of FZL per min at 37 jC. 2.4. Screening of albumin-specific proteinase and detergent Screening of albumin-specific proteinase was performed from commercially available proteinase and thousands of strains of bacteria. A 200 Al of the substrate solution [40 g/l human albumin and 20 g/l human globulins (Cohn Fraction II, III, IV and g-globulins)] was incubated at 37 jC and then the protein digestion reaction was initiated by the addition of 40 Al of proteinase solution. After 30-min incubation at 37 jC, the reaction mixture was filtered through a centrifugal filter unit (Ultrafree MC centrifugal filter unit; 10,000 NMWL). In the case of test blank, the reaction was initiated by adding 40 Al of Tris – HCl buffer (50 mmol/l, pH 8.0) in place of proteinase solution. Then, 300 Al of the
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ketoamine oxidase reagents (50 mmol/l Tris – HCl, pH 8.5, 0.02% 4-aminoantipyrine, 0.02% TODB, 2 U/ml ketoamine oxidase and 5 U/ml peroxidase) were incubated at 37 jC and the reaction was initiated by the addition of 30 Al of proteinase reaction mixture. The absorbance at 555 nm was measured. Screening of the albumin-specific detergent was performed from commercially available detergents. In the assay, 240 Al of the protein digestion reagent (150 mmol/l Tris buffer, pH 8.5, 8.0 mmol/l 4-aminoantipyrine, 15 U/ml peroxidase, 2500 U/ml protease type X X VII and 1% detergents) was incubated at 37 jC with 8.0 Al of substrate solution for 5 min. The absorbance at 546/700 nm was measured. Then, the ketoamine oxidase reaction was initiated by the addition of 80 Al of the ketoamine oxidase reagent (150 mmol/l Tris buffer, pH 8.5, 12 mmol/l TODB, 24 U/ml ketoamine oxidase) and the absorbance at 546/700 nm was measured. The difference between the measurements made before and 5 min after the start of the ketoamine oxidase reaction was calculated. In the case of test blank, protein digestion reagent was incubated at 37 jC with 8.0 Al of Tris – HCl buffer (50 mmol/l, pH 8.5) in place of substrate solution. 2.5. Optimization studies Optimization studies were performed for each of the components of the glycated albumin assay. 2.5.1. Optimum proteinase concentration In the assay, 240 Al of the albumin digestion reagent (50 mmol/l Tris buffer, pH 7.6, 8.0 mmol/l 4-aminoantipyrine and 0 – 10000 U/ml protease type X X VII) was incubated at 37 jC with 8.0 Al of samples (control serum and human serum albumin solution) for 5 min, and the absorbance at 546/700 nm was measured. Then, the ketoamine oxidase reaction was initiated by the addition of 80 Al of the ketoamine oxidase reagent (50 mmol/l Tris buffer, pH 7.5, 12 mmol/l TODB, 15 U/ml peroxidase and 24 U/ml ketoamine oxidase) and the absorbance at 546/700 nm was measured. The difference between the measurements made before and 5 min after the start of the ketoamine oxidase reaction was calculated.
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2.5.2. Optimization of ascorbic acid oxidase concentration of ascorbic acid elimination reaction In the assay, the reagent (50 mmol/l Tris buffer, pH 7.6, 8.0 mmol/l 4-aminoantipyrine, 2500 U/ml protease type X X VII and 1% CHAPSO and 0 – 100 U/ ml ascorbic acid oxidase) was used as the protein digestion reagent. The reaction sequence and ketoamine oxidase reagent composition were the same as described above without proteinase, CHAPSO and ascorbic acid oxidase concentration. 2.5.3. Optimization of ketoamine oxidase concentration In the assay, the reagent (50 mmol/l Tris buffer, pH 7.6, 8.0 mmol/l 4-aminoantipyrine, 2500 U/ml protease type X X VII and 1% CHAPSO) was used as the albumin digestion reagent and the reagent (50 mmol/l Tris buffer, pH 7.5, 12 mmol/l TODB, 15 U/ ml peroxidase and 0– 60 U/ml ketoamine oxidase) was used as the ketoamine oxidase reagent. The reaction sequence was the same as described in Section 2.5.1. 2.6. Assay of glycated albumin The automated enzymatic assay of glycated albumin in biological samples consisted of three steps. Step 1 was assay of glycated albumin concentration using proteinase and ketoamine oxidase; step 2 was assay of albumin concentration; and step 3 was
calculation of percentage of glycated albumin in total albumin (Fig. 1). In the case of using automated analyzer, it takes 10 min to determine glycated albumin with this method. Step 1: In the assay of glycated albumin concentration, 240 Al of the albumin digestion reagent (50 mmol/l Tris buffer, pH 7.6, 6.0 mmol/l 4aminoantipyrine, 2500 U/ml protease type X X VII, 1% CHAPSO and 10 U/ml ascorbic acid oxidase) was incubated at 37 jC with 6.0 Al of samples for 5 min. The absorbance at 546/700 nm was measured. Then, the ketoamine oxidase reaction was initiated by the addition of 80 Al of the ketoamine oxidase reagent (50 mmol/l Tris buffer, pH 7.5, 15 U/ml peroxidase, 10 mmol/l TODB and 24 U/ml ketoamine oxidase) and the absorbance at 546/700 nm was measured. The difference between the measurements made before and 5 min after the start of the ketoamine oxidase reaction was calculated. The concentrations of glycated albumin in specimens were determined by comparison with a calibration curve. The reagents were stable at 4 jC for 2 weeks. Step 2: In the assay of albumin concentration, 270 Al of the albumin assay reagent was incubated at 37 jC with 3.0 Al of samples for 5 min. The absorbance at 660/700 nm was measured. The concentrations of albumin in specimens were determined by comparison with a calibration curve. The reagents were stable at 25 jC for 1 year.
Fig. 1. Assay procedure of an enzymatic method for the measurement of glycated albumin.
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Step 3: The percentage of glycated albumin in total albumin (GA value) was calculated. GA valueð%Þ ¼glycated albumin concentration =albumin concentration 100 2.7. Calibration curves for glycated albumin concentration (g/l) Calibration curves for glycated albumin concentration (g/l) were prepared by plotting the means of absorbance at 546/700 nm (n=2) of the calibrator (normal concentration and abnormal concentration) on the y-axis vs. glycated albumin concentrations (g/ l) on the x-axis. Glycated albumin concentrations of calibrators (g/l) were calculated from the measured glycated amino acid concentration (Amol/l), the measured average number of glycated amino acids per glycated albumin molecule, the glycated amino acid production ratio and theoretical M.W. of native human serum albumin, with the following calculation formula: Glycated albumin ðg=lÞ ¼ measured glycated amino acid concentration ðmol=lÞ=0:8=1:97 66438 where 0.8 is the glycated amino acid production ratio (see Sections 2.7.1 and 3.3), 1.97 is the average number of glycated amino acids per glycated albumin molecule that was measured by enzymatic method (see Sections 2.7.2. and 3.4), and 66438 is the theoretical molecular weight of native human serum albumin [20]. 2.7.1. The glycated amino acid production ratio The glycated amino acid production ratio was determined by changing proteinase reaction time (5 min to 48 h). The reaction sequence and the reagent composition were the same as described in Section 2.6, Step 1. without reaction time. 2.7.2. The average number of glycated amino acids per glycated albumin molecule The average number of glycated amino acids per glycated albumin molecule measured by enzymatic
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method was determined by correlation of GAA value (glycated amino acid/albumin100) measured by enzymatic method and GA value (glycated albumin/albumin 100) measured by the HPLC method (n=70). 2.8. Assay evaluation 2.8.1. Linearity The linearity curve was prepared by plotting the means of GA value (n=3) on the y-axis vs. the mixing ratio of nonglycated albumin (40 g/l: 4.3%) and glycated albumin (40 g/l: 95.4%) on the x-axis. 2.8.2. Dilution test High GA value control for dilution test was prepared two times the usual concentration (glycated albumin concentration: 69.1 g/l; albumin concentration: 85.1 g/l) and diluted. The glycated albumin dilution curve was prepared by plotting the means of glycated albumin concentration of high GA value control (n=3) on the y-axis vs. the dilution rate on the x-axis. The albumin dilution curve was prepared by plotting the means of albumin concentration of high GA value control (n=3) on the y-axis vs. the dilution rate on the x-axis. The dilution curve was prepared by plotting the means of GA value of high GA value control (n=3) on the y-axis vs. the dilution ratio on the xaxis. 2.8.3. Recovery The recovery studies were performed by adding of purified glycated albumin to serum samples. The glycated albumin-added serum samples were prepared by adding 1 volume of glycated albumin solution (0 to 80.0 g/l) to 2 volumes of samples. Serum samples were assayed in five replicates. 2.8.4. Within-run and between-run coefficients of variation (CVs) Serum samples with normal concentrations and abnormal concentrations of glycated albumin were used to determine the within-run and between-run CVs. In a within-run experiment, sample was assayed in 10 replicates. Using the same samples, a between-run experiment was performed on 10 different days.
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2.8.5. Interference studies Interferences from free and conjugated bilirubin, hemoglobin and chyle were determined with the Interference Check-A Plus kit. Various amounts of ascorbic acid and glucose were added to serum at concentrations of 0– 1.00 and 0 –10.0 g/l, respectively, and then the samples were analyzed. Human serum globulins (globulins 1 was prepared from serum collected from healthy donor, globulins 2 was prepared from serum collected from DM patient) and g-globulins were added to serum at final concentrations of about 18 g/l, and then the samples were analyzed. The GA value of serum without interfering substances was measured as a control. 2.8.6. Stability of samples Serum samples with normal concentrations and abnormal concentrations of glycated albumin were used to determine the stability of samples. Samples were stored at 80, 20, 4, 10 or 25 jC for 1 week and assayed in three replicates. 2.8.7. Effects of anticoagulants EDTA2Na (4.5 mg/ml; final concentration), EDTA2K (5.4 mg/ml), heparin (39 U/ml), citric acid (1.14%), oxalic acid (1.2 mg/ml) and an anticoagulants mixtures A (FNa: 1.5 mg/ml; EDTA2Na: 3 mg/l) and B (FNa: 3.75 mg/ml; heparin: 37.5 U/ml; EDTA2Na: 11.1 mg/l) were added to serum and samples were then analyzed. The final concentrations of anticoagulants were three times the usual concentration in commercially available blood collecting tubes. The GA value of serum without anticoagulants was measured as a control. In addition, serum and plasma (EDTA2K, citric acid and heparin) were collected from the same person and the samples were then analyzed. 2.8.8. Investigation of labile intermediate interference One aliquot (100 Al) of each of five diabetic serum containing glucose between 1.41 and 2.88 g/l was dialyzed against 0.9% NaCl and incubated at 4 jC for 12 h to remove glucose. Then, each serum was dialyzed against 0.9% NaCl and incubated 37 jC for 5 h to remove labile intermediate [21]. A second aliquot was kept at 4 jC. Each sample was assayed for glycated albumin.
2.8.9. Correlation between GA value determined by enzymatic method and GA value determined by HPLC method Serum (n=99) and plasma (n=166) were measured by enzymatic method and HPLC method. 2.9. Instrument Automated enzymatic assay of glycated albumin was performed with a Model 7170s Hitachi automatic clinical analyzer (Hitachi, Tokyo, Japan). Measurements of enzyme activity were performed with a Shimazu UV-2100 spectrophotometer with a constant-temperature cell holder. Automated HPLC assay of glycated albumin was performed with a Model HiAuto GAA (GAA-2000) automatic analyzer (Arkray, Kyoto, Japan). 2.10. Statistical analysis Mean, CVs and Pearson’s correlations coefficients (rp) were calculated and linear regression performed using Microsoft Excel 97 (Microsoft). Spearman’s correlations coefficients (rs) were calculated using StatMate (ATMS).
3. Results 3.1. Screening of albumin-specific proteinase and detergent Because protease type X X VII was more specific for albumin than the other proteinase tested, we used it as the albumin-specific proteinase. In addition, since the effect of increasing the albumin specificity of proteinase was checked in CHAPSO, we used CHAPSO as the albumin-specific detergent. 3.2. Optimization studies Since the glycated amino acid production rates from specimens were constant up to a proteinase concentration of 1000 U/ml, we used a concentration of 2500 U/ml for the albumin digestion reagent. In the similar fashion, we used a concentration of ketoamine oxidase 24 U/ml for the ketoamine oxidase reagent.
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It is known that ascorbic acid interferes with detection of H2O2. We, therefore, studied ascorbic acid elimination using ascorbic acid oxidase, which converts ascorbic acid to dehydroascorbic acid. Since rate of the ascorbic acid elimination reaction was constant up to 5 U/ml, we used an ascorbic acid oxidase concentration of 10 U/ml for the protein digestion reagent. 3.3. The glycated amino acid production ratio Maximum glycated amino acid production was measured up to 48 h after starting proteinase reaction. The glycated amino acid production ratios for 5 min to 48 h were about 80%. There were narrow variations in the samples (Table 1). In this paper 0.8 was used as the glycated amino acid production ratio.
Fig. 2. Correlation between GAA value (enzymatic method) and GA value (HPLC method). The GAA value measured by the enzymatic method were significantly correlated with the GA value measured by HPLC method [GAA valueenzymatic method=1.97 (GA valueHPLC method1.46); rp=0.996, rs=0.991].
3.4. The average number of glycated amino acids per glycated albumin molecule 3.5. Assay evaluation GAA values (glycated amino acid/albumin100) measured by enzymatic method were significantly correlated with GA values measured by HPLC method [GAA valueenzymatic method=1.97(GA valueHPLC method1.46), rp=0.996, rs=0.991] (Fig. 2). The average number of glycated amino acids in one molecule of glycated albumin was determined by the slope of correlation curve. In this work, 1.97 was used as the average number of glycated amino acids per glycated albumin molecule.
The linearity curve was linear (r=1.00) between 4.3% and 95.4% (Fig. 3). The glycated albumin dilution curve was linear (r=0.999) between 0.0 and 50.0 g/l (Fig. 4[A]), and the albumin dilution curve was linear (r=0.999) between 0.0 and 60.0 g/l (Fig. 4[B]). Albumin concentration (0.0 –60.0 g/l) did not affect GA value (Fig. 4[C]). Analytical recoveries of exogenous glycated albumin added to serum were 100 –102.5%. The within-
Table 1 Glycated amino acids production ratio Reaction time
Calibrator L Calibrator H Control serum Serum 1 Serum 2 Serum 3 Serum 4 Serum 5 Serum 6 Serum 7 Serum 8 Serum 9 Serum 10
D Absorbance at 546/700 nm:mAbs (GAA production ratio:%) 5 min
6h
24 h
48 h
34.6 (80.5) 117.1 (77.9) 40.7 (77.2) 33.8 (79.3) 44.9 (76.1) 47.1 (80.1) 43.6 (80.0) 40.7 (80.6) 57.5 (80.5) 52.6 (79.3) 93.2 (79.3) 50.4 (81.7) 88.3 (78.7)
42.3 (98.3) 151.1 (100.5) 55.2 (104.7) 36.8 (86.2) 56.7 (96.2) 53.1 (91.4) 50.5 (92.6) 50.1 (99.3) 65.6 (91.9) 63.8 (96.2) 115.0 (97.8) 58.3 (94.6) 105.5 (93.6)
40.8 (94.8) 148.8 (98.9) 51.8 (98.4) 41.1 (96.4) 58.8 (99.8) 56.8 (97.8) 51.5 (94.4) 51.8 (102.6) 71.7 (100.5) 66.0 (99.6) 120.3 (102.3) 60.7 (98.4) 115.6 (102.5)
43.0 (100) 150.4 (100) 52.7 (100) 42.6 (100) 59.0 (100) 58.1 (100) 54.5 (100) 50.5 (100) 71.4 (100) 66.3 (100) 117.6 (100) 61.7 (100) 112.7 (100)
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Fig. 3. Linearity. The linearity curve was prepared by plotting the means of GA value (n=3) on the y-axis vs. the mixing ratio of nonglycated albumin (40 g/l: 4.3%) and glycated albumin (40 g/l: 95.4%) on the x-axis. The linearity curve was linear (r=1.00) between 4.3% and 95.4%.
run (n=10) and between-run (n=10) coefficients of variation were 0.45 –0.67% and 1.1– 1.3%, respectively (data not shown). The addition of up to 200 mg/l (final concentration) of free and conjugated bilirubin, 1500 (turbidity index) of chyle or 10.0 g/l of glucose did not affect the results. Hemoglobin at a concentration of 5.00 g/l caused a 5% negative bias in the assay for glycated albumin. Ascorbic acid at a concentration of 1.00 g/l caused an 8% positive bias in the assay for glycated albumin. The addition of up to 18 g/l (final concentration) of human serum globulins and g-globulins did not affect the results of assay (Fig. 5). Samples with normal concentrations and abnormal concentrations were stable for 1 week at 4 and 10 jC.
Fig. 5. Interference of globulins. Human serum globulins (o, Globulins 1: prepared from the serum collected from healthy donor; ., Globulins 2: prepared from the serum collected from DM patient) were added to serum, and then the sample was analyzed. The addition of up to 18 g/l (final concentration) of human globulins did not affect the results.
At 25 jC, samples were stable for 1 day. Not only plasma that was collected in commercially available blood collecting tubes (anticoagulants: EDTA, heparin and citric acid) but also the addition of anticoagulants at concentrations about three times normal in commercially available blood collecting tubes did not affect the results (data not shown). There was no significant difference in results between pretreated samples removing the labile intermediate and untreated samples. GA values obtained with the present method were significantly correlated with those obtained with HPLC method (serum: GA valueEnzymatic method= 0.979 GA value H P L C m e t h o d 1.14; r p =0.989, rs=0.973, n=99; plasma: GA valueEnzymatic method=
Fig. 4. Dilution test. High-GA value control was prepared two times the usual concentration and diluted. The dilution curve was linear (rp=0.999) between 0.0 and 50.0 g/l [A], and the albumin dilution curve was linear (rp=0.999) between 0.0 and 60.0 g/l [B]. Albumin concentration (0.0 – 60.0 g/l) did not affect GA value [C].
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Ketoamine oxidase has been obtained from such bacterial genera as Corynebacterium [22], Penicillum [23], Aspergillus [24], Pseudomonas [25] and Fusarium [26]. Since the ketoamine oxidase which we used acts not only on q-glycated amino acids but also glycated dipeptides, it is especially useful for measurement of glycated albumin. Enzymatic methods for assaying serum fructosamine using proteinase and ketoamine oxidase have been established [6,7]. An enzymatic method for glycated albumin has not been reported. In order to measure glycated albumin enzymaticaly, we screened albumin-specific proteinase and detergent. We found that protease type X X VII was more specific for albumin than the other proteinase tested. In addition, we found that CHAPSO increased the albumin specificity of proteinase. Since CHAPSO is a derivative of cholic acid, specific binding of CHAPSO to albumin may cause specific denaturation of albumin that then is easily attacked by proteinase. Further research is required to determine why CHAPSO increases the albumin specificity of proteinase. GAA values (glycated amino acid/albumin100) measured by enzymatic method was significantly correlated with GA value measured by HPLC method (rp=0.996, rs=0.992, Fig. 2). GAA valueenzymatic
method
¼ 1:97 ðGA valueHPLC Fig. 6. Correlation between GA value determined by enzymatic method and HPLC method. GA values obtained with the enzymatic method were significantly correlated with those obtained with HPLC method (serum: GA value Enzymatic method=0.979 GA valueHPLC method1.14; rp=0.989, rs=0.973 [A]; plasma: GA value Enzymatic method =0.982 GA value HPLC method 0.99; r p = 0.992, rs=0.987 [B]).
0.982 GA value H PLC method 0.99; r p =0.992, rs=0.987, n=166) (Fig. 6[A], [B]).
4. Discussion In order to determine GA values more easily and rapidly, we developed a new enzymatic method for glycated albumin using albumin-specific proteinase, ketoamine oxidase and albumin assay reagent.
method
1:46Þ
Because the correlation curve was linear and its slope was about 2.0, we speculate that the enzymatic method measures an average of about 2 glycated amino acids per albumin molecule. Our data is in agreement with Guthrow et al. [10] who showed that ‘‘the correlation between determinations of percentage glucosylated albumin by the chemical (thiobarbituric acid) and chromatographic procedure (CM-cellulose) was excellent (GA valuechemical method=1.16GA valuechromatographic method+0.16, r=0.99)’’. In addition, previous reports by Garlic and Mazer [27] showed that ‘‘the nonenzymatic glycation of albumin in vivo occurs at multiple sites’’. Since percentages of glycated amino acid in total albumin measured by not only enzymatic method but also chemical procedure were significantly correlated with GA value measured by chromatographic procedure, we presume that the
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number of glycated amino acids per albumin molecule may be constant regardless of whether healthy individuals or patients with DM are tested. In this study, 1.97 was used as the average number of glycated amino acids preglycated albumin molecule. As for the slope of 1.97, further studies are needed. In addition, since artificially produced control serums are not necessarily equivalent of native serums, the number of glycated amino acids per albumin molecule may be different. Caution is needed concerning the use of artificially produced control serums as calibrator. Since GA value measured by enzymatic method was defined as equal to GAA value/1.97, the correlation curve between GA value measured enzymatic method and HPLC method was showed as follows: GA valueHPLC
method
¼ GAA valueenzymatic ¼ GA valueenzymatic
method =1:97
method
þ 1:46
þ 1:46
Ikeda et al. [15] showed that nonglycated albumin was found to bind to boronate-packed columns in a nonspecific manner. It can be assumed that a yintercept of 1.46 indicates nonspecific binding of nonglycated albumin to columns. As for the y-intercept of 1.46, further studies are needed. The analytical performance (linearity, recovery, within-run and between-run CV) of this method was good with no interference from free and conjugated bilirubin, chyle, glucose and labile intermediate, but with some interference from ascorbic acid and hemoglobin. In order to avoid interference by ascorbic acid, we studied the ascorbic acid elimination reaction using ascorbic acid oxidase. However, ascorbic acid at a concentration of 1.00 g/l (final concentration) caused a positive bias of 8% in the assay for glycated albumin. This appeared to interfere with the detection of H2O2 by dehydroascorbic acid. Since hemoglobin interferes with measurement of glycated albumin, caution is needed concerning the use of hemolyzed samples. In addition, in the case of using automated analyser, it takes 10 min to determine the GA value. This enables a throughput of >800 samples/h, suitable daily clinical use.
In conclusion, this new method is simple, rapid, allows multiple determinations and enables quantitative analysis of GA in biological samples. References [1] Bunn FH, Gabbay HK, Gallop MP. The glycosylation of hemoglobin: relevance to diabetes mellitus. Science 1978; 20:21 – 27. [2] Day JF, Thorpe SR, Baynes J. Non-enzymatic glucosylation of rat serum proteins in vitro and in vivo. Fed Proc 1979; 38:418. [3] Guthrow CE, Morris MA, Day JF, Thorpe SR, Baynes JW. Enhanced nonenzymatic glucosylation of human serum albumin in diabetes mellitus. Proc Natl Acad Sci U S A 1979;76:4258 – 4261. [4] McDonald JM, Davis JE. Glycosylation of serum albumin: elevated glycosyl-albumin in diabetic patients. FEBS Lett 1979;103:282 – 286. [5] Reed P, Bhatnager D, Dhar H, Winocour PH. Precise measurement of glycated serum albumin by column affinity chromatography and immunoturbidmetry. Clin Chim Acta 1986; 161:191 – 199. [6] Koniyama H, Hanada T, Matsuura S. A new enzymatic assay for glycated proteins. Jpn J Clin Chem 1998;27:99 – 106. [7] Moriwaki K, Miyazaki M, Kakutani I, Fujita S, Yasuhara M, Katayama Y. Clinical usefulness of enzymatic assay for fructosamine using protease and fructosylamine oxidase. J Anal Biosci 2000;23:226 – 273. [8] Silver AC, Lamb E, Cattell WR, Dawnay AB. St J Investigation and validation of the affinity chromatography method for measuring glycated albumin in serum and urine. Clin Chim Acta 1991;202:11 – 22. [9] Day JF, Thorpe SR, Baynes JW. Nonenzymatically glucosylated albumin. J Biol Chem 1979;254:595 – 597. [10] Guthrow CE, Morris MA, Day JF, Thorpe SR, Baynes JW. Enhanced nonenzymatic glucosylation of human serum albumin in diabetes mellitus. Proc Natl Acad Sci U S A 1979;76: 4258 – 4261. [11] Dolhofer R, Wieland OH. Improvement of the thiobarbituric acid assay for serum glycosylprotein determination. Clin Chim Acta 1981;112:197 – 204. [12] Kathryn A, Karen JC, Salvatore VP. The standardization of the thiobarbituric acid assay for nonenzymatic glucosylation of human serum albumin. Anal Biochem 1981;118:294 – 300. [13] Ohe Y, Matsuura M, Nakajima Y, Shin S, Hashimoto F, Hirota M, et al. Clin Chim Acta 1987;169:229 – 238. [14] Yamamoto Y, Tahara Y, Cha T, Noma Y, Fukuda M, Yamato E, et al. Radioimmunoassay of glycated serum protein using monoclonal antibody to glucitollysine and coomassie-brilliant-blue-coated polystyrene beads. Diabetes Res. 1989; 11:45 – 49. [15] Ikeda K, Sakamoto Y, Kawasaki Y, Miyake T, Tanaka K, Urata T, et al. Determination of glycated albumin by enzyme linked boronate immunoassay (ELBIA). Clin Chem 1998;44: 256 – 263.
T. Kouzuma et al. / Clinica Chimica Acta 324 (2002) 61–71 [16] Shima K, Ito N, Abe F, Yano M, Yamamoto Y, Uchida T, et al. High-performance liquid chromatographic assay of serum glycated albumin. Diabetologia 1988;31:627 – 631. [17] Yasukawa K, Abe F, Shida N, Koizumi Y, Uchida T, Noguchi K, et al. High-performance affinity chromatography system for the rapid, efficient assay of glycated albumin. J Chromatogr 1992;597:271 – 275. [18] Hashiba H. Participation of amadori rearrangement products and carbonyl compounds in oxygen-dependent browning of soy sauce. J Agric Food Chem 1976;24:70 – 73. [19] Matsuda N, Takahashi H. Standards for quantitative analysis of human serum albumin. Official J Jpn Soc Lab Med 1975;23:889 – 894. [20] Geisow MJ, Harris R, Dodsworth N, Green B, Hutton T. Characterization and quality assurance of recombinant human serum albumin. In: Villafranca JJ, editor. Techniques in protein chemistry. San Diego, CA: Academic Press; 1991. p. 572 – 576. [21] Goldstein DE, Peth SB, England JD, Hess RL, Costa JD. Effects of acute changes in blood glucose on HbA1c. Diabetes 1980;29:623 – 628.
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[22] Horiuchi T, Kurokawa T, Saito N. Purification and properties of fructosyl-amino acid oxidase from Corynebacterium sp. 24-1. Agric Biol Chem 1989;53(1):103 – 110. [23] Watanabe N, Ohtsuka M, Takahashi S, Sakano Y, Fujimoto D. Enzymatic deglycation of fructosyl-lysine. Agric Biol Chem 1990;54(4):1063 – 1064. [24] Horiuchi T, Kurokawa T. Purification and properties of fructosylamine oxidase from Aspergillus sp. Agric Biol Chem 1991;55(2):333 – 338. [25] Gerhardinger C, Marion MS, Rover A, Glomb M, Monnier VM. Novel degradation pathway of glycated amino acids into free fructosamine by Pseudomonas sp. soil extract. J Biol Chem 1995;270(1):218 – 224. [26] Yoshida N, Sakai Y, Serata M, Tani Y, Kato N. Distribution and properties of fructosyl amino acid oxidase in fungi. Appl Environ Microbiol 1995;61(12):4487 – 4489. [27] Garlic RL, Mazer JS. The principal site of nonenzymatic glycosylation of human serum albumin in vivo. J Biol Chem 1983;258(10):6142 – 6146.
An enzymatic method for the measurement of glycated albumin in biological samples Takuji Kouzuma*, Tomomi Usami, Masaru Yamakoshi, Mamoru Takahashi, Shigeyuki Imamura Diagnostics R&D Department, Fine Chemicals and Diagnostics Division, Health Care Company, Asahi Kasei Corporation. 632-1, Mifuku, Ohito-cho, Tagata-gun, Shizuoka-ken, 410-2321, Japan Received 8 April 2002; received in revised form 10 June 2002; accepted 10 June 2002
Abstract Background: In order to determine glycated albumin more easily and rapidly, we developed a new enzymatic method for glycated albumin in blood samples. Methods: The method involves use of albumin-specific proteinase, ketoamine oxidase and serum albumin assay reagent. In the assay, glycated albumin is hydrolyzed to glycated amino acids by proteinase digestion, and ketoamine oxidase oxidizes the glycated amino acids to produce hydrogen peroxide, which is quantitatively measured. Glycated albumin is calculated as the percentage of glycated albumin in total albumin. Results: The calibration curve for glycated albumin concentration was linear (rp=0.999) between 0.0 and 50.0 g/l and that for albumin concentration was linear (rp=0.999) between 0.0 and 60.0 g/l. The analytical recoveries of exogenous glycated albumin added to serum were 100 – 102.5%. The within-run and between-run CVs were 0.45 – 0.67% and 1.09 – 1.26%, respectively. This method was free from interference by bilirubin, chyle, glucose, globulins and labile intermediate. Weak interference by hemoglobin and ascorbic acid was observed. Glycated albumin detected by the present method was significantly correlated with glycated albumin detected by highperformance liquid-chromatographic (HPLC) method (serum: rs=0.989, plasma: rp=0.992). Conclusions: This new enzymatic method is simple, rapid, allows multiple determinations and enables quantitative analysis of glycated albumin. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Glycated albumin; Proteinase; Ketoamine oxidase
1. Introduction Chronic hyperglycemia in diabetes results in increased concentrations of nonenzymatically glycated proteins including hemoglobin [1] and albumin [2,3]. Since the modification of hemoglobin by glucose occurs continually during the life span of * Corresponding author. Tel.: +81-0558-76-8607; fax: +810558-76-7149. E-mail address: [email protected] (T. Kouzuma).
the erythrocyte, glycated hemoglobin concentrations provide a time-averaged index of the degree of hyperglycemia during the previous 2 months in humans [4]. In the same way, glycated albumin appears to provide an index of the state of glycemic control for approximately the previous 2 weeks. The concentration of glycated albumin should provide additional useful information on glycemic control when monitoring effects of changes in diet or insulin therapy. The serum concentration of fructosamine may also be used as an index of glycemic control for the
0009-8981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 8 9 8 1 ( 0 2 ) 0 0 2 0 7 - 3
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previous 2 weeks, but fructosamine concentrations are strongly influenced by the concentrations of protein, bilirubin, hemoglobin, uric acid and other low-molecular-weight substances coexisting in the blood and are, therefore, less accurate [5]. In order to avoid effects of low-molecular-weight substances coexisting in the blood, an enzymatic assay for fructosamine using proteinase and ketoamine oxidase [6,7] was developed. However, since fructosamine concentrations determined by the enzymatic method are strongly influenced by concentration of protein and the exact half-lives of all glycated proteins have not yet been determined, measurement of glycated albumin may be more useful clinically. The concentrations of glycated albumin in biological specimens have been measured by affinity chromatography [5,8], ion exchange chromatography [9,10], thiobarbituric acid assay [11,12], radioimmunoassay [13,14] and boronate immunoassay [15]. However, these methods have a number of disadvantages, e.g. specimens must be pretreated and procedures are complicated. A simple high-performance liquid-chromatographic (HPLC) method for separation of glycated albumin involving a combination of ion-exchange chromatography to separate albumin and boronate affinity chromatography to separate glycated albumin from nonglycated albumin [16,17] has been established. However, this method could measure only 12 samples/h, a rate not clearly sufficient for routine clinical use. An enzymatic method for glycated albumin has not been reported. In order to determine glycated albumin more easily and rapidly, we developed a new enzymatic method for glycated albumin using albumin-specific proteinase, ketoamine oxidase and albumin assay reagent. We describe the screening of albumin-specific proteinase and detergent, optimization studies and the assay evaluation of this enzymatic method.
2. Materials and methods 2.1. Materials Common reagents, albumin assay reagent and fructosamine assay reagent were from Wako (Osaka, Japan). Ascorbic acid oxidase (cucurbita species)
was from Roche Diagnostics (Mannheim, Germany). Folin and Ciocalteu’s phenol reagent, albumin (human; essentially globulin-free), globulins (human; Cohn Fraction II, III, IV and g-globulins), proteinase (protease type X X VUU) and peroxidase (Type II from horseradish) were from Sigma (St. Louis, MO). Interference Check-A Plus (free and conjugated bilirubin, hemoglobin and chyle) was from Kokusai Shiyaku, (Kobe, Japan). N,N-Bis(4-sulfobutyl)-3methylanilin, disodium salt (TODB) and 3-[(3-Cholamido-propyl) dimethyl-ammonio]-2-hydroxypropanesulfonic acid (CHAPSO), were from Dojindo Laboratories (Kumamoto, Japan). Ultrafree MC centrifugal filter unit was from Millipore (Bedford, MA). Fresh, frozen human plasma was from Veritas (Tokyo, Japan). Ketoamine oxidase was from Asahi Kasei (Tokyo, Japan). Blood samples were separated by centrifugation at 3000g at 4 jC. All samples were collected as spot samples and stored at 80 jC without antiseptics until assay. 2.2. Substrates q-N-(1-deoxy-D-fructosyl)-a-Z-lysine (FZL) was, essentially, prepared by the method of Hashiba [18]. Human serum globulins were prepared by the method of Shima et al. [16]. Globulins were separate from albumin by anion exchange column. Nonglycated albumin and glycated albumin were purified from fresh, frozen human plasma. First, human serum albumin was prepared by the method of Matsuda and Takahashi [19]. Then, 1.0 g of human serum albumin was dissolved in 100 ml of 0.05 mol/l glycine – NaOH containing 20 g/l MgCl2 (pH 8.5) and incubated with stirring for 0.5 h at 4 jC with 70 ml of phenylboronic acid resin equilibrated with the same buffer. The solution was passed through a glass filter and the filtrate was pooled as a nonglycated albumin solution. Phenylboronic acid resin was suspended in 30 ml of 0.02 mol/l Tris(hydroxymethyl)aminomethane (Tris) buffer containing 150 mmol/l sorbitol and 440 mmol/l NaCl (pH 8.5) and incubated with stirring for 0.5 h at 4 jC The solution was passed through a glass filter and the filtrate was pooled as a glycated albumin solution. The glycated albumin solution and nonglycated albumin solution were then repurified in the same manner. The final
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solution obtained was concentrated to 40 g/l by an ultrafiltration system. The purified nonglycated albumin solution and glycated albumin solution were assayed with a fructosamine assay kit and HPLC method (GA value of nonglycated albumin solution and glycated albumin solution were 4.3% and 95.4%, respectively). A high GA value control was prepared by incubation of fresh, frozen human plasma with 400 mmol/l glucose (100 mmol/l phosphate buffer pH 7.5) at 37 jC for 14 days. The reaction mixture was dialyzed against 20 mmol/l Tris – HCl buffer (pH 7.5) and lyophilized. 2.3. Definition of proteinase and ketoamine oxidase activity 2.3.1. Proteinase activity The proteinase activity was determined by caseinFolin method. One unit (1U) of proteinase was defined as the amount of enzyme that catalyzed casein to produce color equivalent to 1.0 Ag of tyrosine per minute at 30 jC (color by Folin and Ciocalteu’s phenol reagent). 2.3.2. Ketoamine oxidase activity Ketoamine oxidase activity was monitored by the release of H2O2. One unit (1U) of ketoamine oxidase was defined as the amount of enzyme that oxidizes 1 Amol of FZL per min at 37 jC. 2.4. Screening of albumin-specific proteinase and detergent Screening of albumin-specific proteinase was performed from commercially available proteinase and thousands of strains of bacteria. A 200 Al of the substrate solution [40 g/l human albumin and 20 g/l human globulins (Cohn Fraction II, III, IV and g-globulins)] was incubated at 37 jC and then the protein digestion reaction was initiated by the addition of 40 Al of proteinase solution. After 30-min incubation at 37 jC, the reaction mixture was filtered through a centrifugal filter unit (Ultrafree MC centrifugal filter unit; 10,000 NMWL). In the case of test blank, the reaction was initiated by adding 40 Al of Tris – HCl buffer (50 mmol/l, pH 8.0) in place of proteinase solution. Then, 300 Al of the
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ketoamine oxidase reagents (50 mmol/l Tris – HCl, pH 8.5, 0.02% 4-aminoantipyrine, 0.02% TODB, 2 U/ml ketoamine oxidase and 5 U/ml peroxidase) were incubated at 37 jC and the reaction was initiated by the addition of 30 Al of proteinase reaction mixture. The absorbance at 555 nm was measured. Screening of the albumin-specific detergent was performed from commercially available detergents. In the assay, 240 Al of the protein digestion reagent (150 mmol/l Tris buffer, pH 8.5, 8.0 mmol/l 4-aminoantipyrine, 15 U/ml peroxidase, 2500 U/ml protease type X X VII and 1% detergents) was incubated at 37 jC with 8.0 Al of substrate solution for 5 min. The absorbance at 546/700 nm was measured. Then, the ketoamine oxidase reaction was initiated by the addition of 80 Al of the ketoamine oxidase reagent (150 mmol/l Tris buffer, pH 8.5, 12 mmol/l TODB, 24 U/ml ketoamine oxidase) and the absorbance at 546/700 nm was measured. The difference between the measurements made before and 5 min after the start of the ketoamine oxidase reaction was calculated. In the case of test blank, protein digestion reagent was incubated at 37 jC with 8.0 Al of Tris – HCl buffer (50 mmol/l, pH 8.5) in place of substrate solution. 2.5. Optimization studies Optimization studies were performed for each of the components of the glycated albumin assay. 2.5.1. Optimum proteinase concentration In the assay, 240 Al of the albumin digestion reagent (50 mmol/l Tris buffer, pH 7.6, 8.0 mmol/l 4-aminoantipyrine and 0 – 10000 U/ml protease type X X VII) was incubated at 37 jC with 8.0 Al of samples (control serum and human serum albumin solution) for 5 min, and the absorbance at 546/700 nm was measured. Then, the ketoamine oxidase reaction was initiated by the addition of 80 Al of the ketoamine oxidase reagent (50 mmol/l Tris buffer, pH 7.5, 12 mmol/l TODB, 15 U/ml peroxidase and 24 U/ml ketoamine oxidase) and the absorbance at 546/700 nm was measured. The difference between the measurements made before and 5 min after the start of the ketoamine oxidase reaction was calculated.
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2.5.2. Optimization of ascorbic acid oxidase concentration of ascorbic acid elimination reaction In the assay, the reagent (50 mmol/l Tris buffer, pH 7.6, 8.0 mmol/l 4-aminoantipyrine, 2500 U/ml protease type X X VII and 1% CHAPSO and 0 – 100 U/ ml ascorbic acid oxidase) was used as the protein digestion reagent. The reaction sequence and ketoamine oxidase reagent composition were the same as described above without proteinase, CHAPSO and ascorbic acid oxidase concentration. 2.5.3. Optimization of ketoamine oxidase concentration In the assay, the reagent (50 mmol/l Tris buffer, pH 7.6, 8.0 mmol/l 4-aminoantipyrine, 2500 U/ml protease type X X VII and 1% CHAPSO) was used as the albumin digestion reagent and the reagent (50 mmol/l Tris buffer, pH 7.5, 12 mmol/l TODB, 15 U/ ml peroxidase and 0– 60 U/ml ketoamine oxidase) was used as the ketoamine oxidase reagent. The reaction sequence was the same as described in Section 2.5.1. 2.6. Assay of glycated albumin The automated enzymatic assay of glycated albumin in biological samples consisted of three steps. Step 1 was assay of glycated albumin concentration using proteinase and ketoamine oxidase; step 2 was assay of albumin concentration; and step 3 was
calculation of percentage of glycated albumin in total albumin (Fig. 1). In the case of using automated analyzer, it takes 10 min to determine glycated albumin with this method. Step 1: In the assay of glycated albumin concentration, 240 Al of the albumin digestion reagent (50 mmol/l Tris buffer, pH 7.6, 6.0 mmol/l 4aminoantipyrine, 2500 U/ml protease type X X VII, 1% CHAPSO and 10 U/ml ascorbic acid oxidase) was incubated at 37 jC with 6.0 Al of samples for 5 min. The absorbance at 546/700 nm was measured. Then, the ketoamine oxidase reaction was initiated by the addition of 80 Al of the ketoamine oxidase reagent (50 mmol/l Tris buffer, pH 7.5, 15 U/ml peroxidase, 10 mmol/l TODB and 24 U/ml ketoamine oxidase) and the absorbance at 546/700 nm was measured. The difference between the measurements made before and 5 min after the start of the ketoamine oxidase reaction was calculated. The concentrations of glycated albumin in specimens were determined by comparison with a calibration curve. The reagents were stable at 4 jC for 2 weeks. Step 2: In the assay of albumin concentration, 270 Al of the albumin assay reagent was incubated at 37 jC with 3.0 Al of samples for 5 min. The absorbance at 660/700 nm was measured. The concentrations of albumin in specimens were determined by comparison with a calibration curve. The reagents were stable at 25 jC for 1 year.
Fig. 1. Assay procedure of an enzymatic method for the measurement of glycated albumin.
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Step 3: The percentage of glycated albumin in total albumin (GA value) was calculated. GA valueð%Þ ¼glycated albumin concentration =albumin concentration 100 2.7. Calibration curves for glycated albumin concentration (g/l) Calibration curves for glycated albumin concentration (g/l) were prepared by plotting the means of absorbance at 546/700 nm (n=2) of the calibrator (normal concentration and abnormal concentration) on the y-axis vs. glycated albumin concentrations (g/ l) on the x-axis. Glycated albumin concentrations of calibrators (g/l) were calculated from the measured glycated amino acid concentration (Amol/l), the measured average number of glycated amino acids per glycated albumin molecule, the glycated amino acid production ratio and theoretical M.W. of native human serum albumin, with the following calculation formula: Glycated albumin ðg=lÞ ¼ measured glycated amino acid concentration ðmol=lÞ=0:8=1:97 66438 where 0.8 is the glycated amino acid production ratio (see Sections 2.7.1 and 3.3), 1.97 is the average number of glycated amino acids per glycated albumin molecule that was measured by enzymatic method (see Sections 2.7.2. and 3.4), and 66438 is the theoretical molecular weight of native human serum albumin [20]. 2.7.1. The glycated amino acid production ratio The glycated amino acid production ratio was determined by changing proteinase reaction time (5 min to 48 h). The reaction sequence and the reagent composition were the same as described in Section 2.6, Step 1. without reaction time. 2.7.2. The average number of glycated amino acids per glycated albumin molecule The average number of glycated amino acids per glycated albumin molecule measured by enzymatic
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method was determined by correlation of GAA value (glycated amino acid/albumin100) measured by enzymatic method and GA value (glycated albumin/albumin 100) measured by the HPLC method (n=70). 2.8. Assay evaluation 2.8.1. Linearity The linearity curve was prepared by plotting the means of GA value (n=3) on the y-axis vs. the mixing ratio of nonglycated albumin (40 g/l: 4.3%) and glycated albumin (40 g/l: 95.4%) on the x-axis. 2.8.2. Dilution test High GA value control for dilution test was prepared two times the usual concentration (glycated albumin concentration: 69.1 g/l; albumin concentration: 85.1 g/l) and diluted. The glycated albumin dilution curve was prepared by plotting the means of glycated albumin concentration of high GA value control (n=3) on the y-axis vs. the dilution rate on the x-axis. The albumin dilution curve was prepared by plotting the means of albumin concentration of high GA value control (n=3) on the y-axis vs. the dilution rate on the x-axis. The dilution curve was prepared by plotting the means of GA value of high GA value control (n=3) on the y-axis vs. the dilution ratio on the xaxis. 2.8.3. Recovery The recovery studies were performed by adding of purified glycated albumin to serum samples. The glycated albumin-added serum samples were prepared by adding 1 volume of glycated albumin solution (0 to 80.0 g/l) to 2 volumes of samples. Serum samples were assayed in five replicates. 2.8.4. Within-run and between-run coefficients of variation (CVs) Serum samples with normal concentrations and abnormal concentrations of glycated albumin were used to determine the within-run and between-run CVs. In a within-run experiment, sample was assayed in 10 replicates. Using the same samples, a between-run experiment was performed on 10 different days.
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2.8.5. Interference studies Interferences from free and conjugated bilirubin, hemoglobin and chyle were determined with the Interference Check-A Plus kit. Various amounts of ascorbic acid and glucose were added to serum at concentrations of 0– 1.00 and 0 –10.0 g/l, respectively, and then the samples were analyzed. Human serum globulins (globulins 1 was prepared from serum collected from healthy donor, globulins 2 was prepared from serum collected from DM patient) and g-globulins were added to serum at final concentrations of about 18 g/l, and then the samples were analyzed. The GA value of serum without interfering substances was measured as a control. 2.8.6. Stability of samples Serum samples with normal concentrations and abnormal concentrations of glycated albumin were used to determine the stability of samples. Samples were stored at 80, 20, 4, 10 or 25 jC for 1 week and assayed in three replicates. 2.8.7. Effects of anticoagulants EDTA2Na (4.5 mg/ml; final concentration), EDTA2K (5.4 mg/ml), heparin (39 U/ml), citric acid (1.14%), oxalic acid (1.2 mg/ml) and an anticoagulants mixtures A (FNa: 1.5 mg/ml; EDTA2Na: 3 mg/l) and B (FNa: 3.75 mg/ml; heparin: 37.5 U/ml; EDTA2Na: 11.1 mg/l) were added to serum and samples were then analyzed. The final concentrations of anticoagulants were three times the usual concentration in commercially available blood collecting tubes. The GA value of serum without anticoagulants was measured as a control. In addition, serum and plasma (EDTA2K, citric acid and heparin) were collected from the same person and the samples were then analyzed. 2.8.8. Investigation of labile intermediate interference One aliquot (100 Al) of each of five diabetic serum containing glucose between 1.41 and 2.88 g/l was dialyzed against 0.9% NaCl and incubated at 4 jC for 12 h to remove glucose. Then, each serum was dialyzed against 0.9% NaCl and incubated 37 jC for 5 h to remove labile intermediate [21]. A second aliquot was kept at 4 jC. Each sample was assayed for glycated albumin.
2.8.9. Correlation between GA value determined by enzymatic method and GA value determined by HPLC method Serum (n=99) and plasma (n=166) were measured by enzymatic method and HPLC method. 2.9. Instrument Automated enzymatic assay of glycated albumin was performed with a Model 7170s Hitachi automatic clinical analyzer (Hitachi, Tokyo, Japan). Measurements of enzyme activity were performed with a Shimazu UV-2100 spectrophotometer with a constant-temperature cell holder. Automated HPLC assay of glycated albumin was performed with a Model HiAuto GAA (GAA-2000) automatic analyzer (Arkray, Kyoto, Japan). 2.10. Statistical analysis Mean, CVs and Pearson’s correlations coefficients (rp) were calculated and linear regression performed using Microsoft Excel 97 (Microsoft). Spearman’s correlations coefficients (rs) were calculated using StatMate (ATMS).
3. Results 3.1. Screening of albumin-specific proteinase and detergent Because protease type X X VII was more specific for albumin than the other proteinase tested, we used it as the albumin-specific proteinase. In addition, since the effect of increasing the albumin specificity of proteinase was checked in CHAPSO, we used CHAPSO as the albumin-specific detergent. 3.2. Optimization studies Since the glycated amino acid production rates from specimens were constant up to a proteinase concentration of 1000 U/ml, we used a concentration of 2500 U/ml for the albumin digestion reagent. In the similar fashion, we used a concentration of ketoamine oxidase 24 U/ml for the ketoamine oxidase reagent.
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It is known that ascorbic acid interferes with detection of H2O2. We, therefore, studied ascorbic acid elimination using ascorbic acid oxidase, which converts ascorbic acid to dehydroascorbic acid. Since rate of the ascorbic acid elimination reaction was constant up to 5 U/ml, we used an ascorbic acid oxidase concentration of 10 U/ml for the protein digestion reagent. 3.3. The glycated amino acid production ratio Maximum glycated amino acid production was measured up to 48 h after starting proteinase reaction. The glycated amino acid production ratios for 5 min to 48 h were about 80%. There were narrow variations in the samples (Table 1). In this paper 0.8 was used as the glycated amino acid production ratio.
Fig. 2. Correlation between GAA value (enzymatic method) and GA value (HPLC method). The GAA value measured by the enzymatic method were significantly correlated with the GA value measured by HPLC method [GAA valueenzymatic method=1.97 (GA valueHPLC method1.46); rp=0.996, rs=0.991].
3.4. The average number of glycated amino acids per glycated albumin molecule 3.5. Assay evaluation GAA values (glycated amino acid/albumin100) measured by enzymatic method were significantly correlated with GA values measured by HPLC method [GAA valueenzymatic method=1.97(GA valueHPLC method1.46), rp=0.996, rs=0.991] (Fig. 2). The average number of glycated amino acids in one molecule of glycated albumin was determined by the slope of correlation curve. In this work, 1.97 was used as the average number of glycated amino acids per glycated albumin molecule.
The linearity curve was linear (r=1.00) between 4.3% and 95.4% (Fig. 3). The glycated albumin dilution curve was linear (r=0.999) between 0.0 and 50.0 g/l (Fig. 4[A]), and the albumin dilution curve was linear (r=0.999) between 0.0 and 60.0 g/l (Fig. 4[B]). Albumin concentration (0.0 –60.0 g/l) did not affect GA value (Fig. 4[C]). Analytical recoveries of exogenous glycated albumin added to serum were 100 –102.5%. The within-
Table 1 Glycated amino acids production ratio Reaction time
Calibrator L Calibrator H Control serum Serum 1 Serum 2 Serum 3 Serum 4 Serum 5 Serum 6 Serum 7 Serum 8 Serum 9 Serum 10
D Absorbance at 546/700 nm:mAbs (GAA production ratio:%) 5 min
6h
24 h
48 h
34.6 (80.5) 117.1 (77.9) 40.7 (77.2) 33.8 (79.3) 44.9 (76.1) 47.1 (80.1) 43.6 (80.0) 40.7 (80.6) 57.5 (80.5) 52.6 (79.3) 93.2 (79.3) 50.4 (81.7) 88.3 (78.7)
42.3 (98.3) 151.1 (100.5) 55.2 (104.7) 36.8 (86.2) 56.7 (96.2) 53.1 (91.4) 50.5 (92.6) 50.1 (99.3) 65.6 (91.9) 63.8 (96.2) 115.0 (97.8) 58.3 (94.6) 105.5 (93.6)
40.8 (94.8) 148.8 (98.9) 51.8 (98.4) 41.1 (96.4) 58.8 (99.8) 56.8 (97.8) 51.5 (94.4) 51.8 (102.6) 71.7 (100.5) 66.0 (99.6) 120.3 (102.3) 60.7 (98.4) 115.6 (102.5)
43.0 (100) 150.4 (100) 52.7 (100) 42.6 (100) 59.0 (100) 58.1 (100) 54.5 (100) 50.5 (100) 71.4 (100) 66.3 (100) 117.6 (100) 61.7 (100) 112.7 (100)
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Fig. 3. Linearity. The linearity curve was prepared by plotting the means of GA value (n=3) on the y-axis vs. the mixing ratio of nonglycated albumin (40 g/l: 4.3%) and glycated albumin (40 g/l: 95.4%) on the x-axis. The linearity curve was linear (r=1.00) between 4.3% and 95.4%.
run (n=10) and between-run (n=10) coefficients of variation were 0.45 –0.67% and 1.1– 1.3%, respectively (data not shown). The addition of up to 200 mg/l (final concentration) of free and conjugated bilirubin, 1500 (turbidity index) of chyle or 10.0 g/l of glucose did not affect the results. Hemoglobin at a concentration of 5.00 g/l caused a 5% negative bias in the assay for glycated albumin. Ascorbic acid at a concentration of 1.00 g/l caused an 8% positive bias in the assay for glycated albumin. The addition of up to 18 g/l (final concentration) of human serum globulins and g-globulins did not affect the results of assay (Fig. 5). Samples with normal concentrations and abnormal concentrations were stable for 1 week at 4 and 10 jC.
Fig. 5. Interference of globulins. Human serum globulins (o, Globulins 1: prepared from the serum collected from healthy donor; ., Globulins 2: prepared from the serum collected from DM patient) were added to serum, and then the sample was analyzed. The addition of up to 18 g/l (final concentration) of human globulins did not affect the results.
At 25 jC, samples were stable for 1 day. Not only plasma that was collected in commercially available blood collecting tubes (anticoagulants: EDTA, heparin and citric acid) but also the addition of anticoagulants at concentrations about three times normal in commercially available blood collecting tubes did not affect the results (data not shown). There was no significant difference in results between pretreated samples removing the labile intermediate and untreated samples. GA values obtained with the present method were significantly correlated with those obtained with HPLC method (serum: GA valueEnzymatic method= 0.979 GA value H P L C m e t h o d 1.14; r p =0.989, rs=0.973, n=99; plasma: GA valueEnzymatic method=
Fig. 4. Dilution test. High-GA value control was prepared two times the usual concentration and diluted. The dilution curve was linear (rp=0.999) between 0.0 and 50.0 g/l [A], and the albumin dilution curve was linear (rp=0.999) between 0.0 and 60.0 g/l [B]. Albumin concentration (0.0 – 60.0 g/l) did not affect GA value [C].
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Ketoamine oxidase has been obtained from such bacterial genera as Corynebacterium [22], Penicillum [23], Aspergillus [24], Pseudomonas [25] and Fusarium [26]. Since the ketoamine oxidase which we used acts not only on q-glycated amino acids but also glycated dipeptides, it is especially useful for measurement of glycated albumin. Enzymatic methods for assaying serum fructosamine using proteinase and ketoamine oxidase have been established [6,7]. An enzymatic method for glycated albumin has not been reported. In order to measure glycated albumin enzymaticaly, we screened albumin-specific proteinase and detergent. We found that protease type X X VII was more specific for albumin than the other proteinase tested. In addition, we found that CHAPSO increased the albumin specificity of proteinase. Since CHAPSO is a derivative of cholic acid, specific binding of CHAPSO to albumin may cause specific denaturation of albumin that then is easily attacked by proteinase. Further research is required to determine why CHAPSO increases the albumin specificity of proteinase. GAA values (glycated amino acid/albumin100) measured by enzymatic method was significantly correlated with GA value measured by HPLC method (rp=0.996, rs=0.992, Fig. 2). GAA valueenzymatic
method
¼ 1:97 ðGA valueHPLC Fig. 6. Correlation between GA value determined by enzymatic method and HPLC method. GA values obtained with the enzymatic method were significantly correlated with those obtained with HPLC method (serum: GA value Enzymatic method=0.979 GA valueHPLC method1.14; rp=0.989, rs=0.973 [A]; plasma: GA value Enzymatic method =0.982 GA value HPLC method 0.99; r p = 0.992, rs=0.987 [B]).
0.982 GA value H PLC method 0.99; r p =0.992, rs=0.987, n=166) (Fig. 6[A], [B]).
4. Discussion In order to determine GA values more easily and rapidly, we developed a new enzymatic method for glycated albumin using albumin-specific proteinase, ketoamine oxidase and albumin assay reagent.
method
1:46Þ
Because the correlation curve was linear and its slope was about 2.0, we speculate that the enzymatic method measures an average of about 2 glycated amino acids per albumin molecule. Our data is in agreement with Guthrow et al. [10] who showed that ‘‘the correlation between determinations of percentage glucosylated albumin by the chemical (thiobarbituric acid) and chromatographic procedure (CM-cellulose) was excellent (GA valuechemical method=1.16GA valuechromatographic method+0.16, r=0.99)’’. In addition, previous reports by Garlic and Mazer [27] showed that ‘‘the nonenzymatic glycation of albumin in vivo occurs at multiple sites’’. Since percentages of glycated amino acid in total albumin measured by not only enzymatic method but also chemical procedure were significantly correlated with GA value measured by chromatographic procedure, we presume that the
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number of glycated amino acids per albumin molecule may be constant regardless of whether healthy individuals or patients with DM are tested. In this study, 1.97 was used as the average number of glycated amino acids preglycated albumin molecule. As for the slope of 1.97, further studies are needed. In addition, since artificially produced control serums are not necessarily equivalent of native serums, the number of glycated amino acids per albumin molecule may be different. Caution is needed concerning the use of artificially produced control serums as calibrator. Since GA value measured by enzymatic method was defined as equal to GAA value/1.97, the correlation curve between GA value measured enzymatic method and HPLC method was showed as follows: GA valueHPLC
method
¼ GAA valueenzymatic ¼ GA valueenzymatic
method =1:97
method
þ 1:46
þ 1:46
Ikeda et al. [15] showed that nonglycated albumin was found to bind to boronate-packed columns in a nonspecific manner. It can be assumed that a yintercept of 1.46 indicates nonspecific binding of nonglycated albumin to columns. As for the y-intercept of 1.46, further studies are needed. The analytical performance (linearity, recovery, within-run and between-run CV) of this method was good with no interference from free and conjugated bilirubin, chyle, glucose and labile intermediate, but with some interference from ascorbic acid and hemoglobin. In order to avoid interference by ascorbic acid, we studied the ascorbic acid elimination reaction using ascorbic acid oxidase. However, ascorbic acid at a concentration of 1.00 g/l (final concentration) caused a positive bias of 8% in the assay for glycated albumin. This appeared to interfere with the detection of H2O2 by dehydroascorbic acid. Since hemoglobin interferes with measurement of glycated albumin, caution is needed concerning the use of hemolyzed samples. In addition, in the case of using automated analyser, it takes 10 min to determine the GA value. This enables a throughput of >800 samples/h, suitable daily clinical use.
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