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A specific method of glycosylated hemoglobin using furosine measurement
Diabetes Research and Clinical Practice, 1 (1985) 193-195
Elsevier
193
DRC00030
A specific method of glycosylated hemoglobin using furosine measurement M u n e t a d a Oimomi 1, Shigeki Nishimoto, Shinichiro M a t s u m o t o , Hiroshi H a t a n a k a , K a z u o Ishikawa and Shigeaki Baba The Second Department of hzternal Medicine, Kobe University School of Medicine, Chuo-ku, Kobe, 650, Japan
(Received 22 February 1985,accepted 26 July 1985)
Key words: Glycosylatedhemoglobin;Fructose-lysine;Furosine; Diabetes mellitus
Summary In order to get an accurately specific determination of glycosylated hemoglobin (GHb-t), the furosine assay method, which can detect e-amino-lysine-bound glucose, was investigated. A good correlation was found between GHb-f values obtained by this method and HbA~ values by conventional ion exchange column chromatography. It was, therefore, considered that this technique would be a new useful method for determining glycosylated hemoglobin.
Introduction Recently it has become feasible to determine glycosylation of protein by measuring furosine derived from fructose-lysine, glucose bound to lysine residues of protein, by acid hydrolysis [1]. In the present study we investigated glycosylation of globin, a protein of hemoglobin (Hb).
Subjects and Methods Seven normal subjects and 44 diabetic patients were Correspondence to: Munetada Oimomi, M.D., The Second Department of Internal Medicine,Kobe UniversitySchool of Medicine, 5-2, 7-chome, Kusunoki-cho,Chuo-ku, Kobe, 650, Japan.
studied. Each blood sample collected in a heparinized syringe was determined for HbAI by high-performance liquid chromatography (HPLC, Auto HbAlc, Kyoto Daiichi Co.) using an ion exchange column method, and for glycosylated Hb by the furosine determination method (glycosylated Hb determined by furosine assay is referred to as GHb0. In determining stable HbA1, the method of Nathan et al. [2] was used. For the determination of GHb-f, globin was extracted with acid acetone from 500 #1 of blood and hydrolyzed with 2 ml of 6 N HCI for 30 h at 95°C [3]. After evaporation to dryness, 500/d of distilled water was added for reconstitution and the resultant solution was used as a sample. In the analysis of furosine, HPLC (Waters) and a TSK-gel column (ODS-120T, Toyo Soda Co.) were employed and 7 mM phosphate was used
0168-8227/85/$03.30 © 1985 ElsevierSciencePublishers B.V. (BiomedicalDivision)
194 TABLE 1 C O R R E L A T I O N BETWEEN G H b - f A N D HbA~ C O M P O N E N T S IN D I A B E T I C P A T I E N T S G H b - f represents the glycosylated hemoglobin determined by furosine measurement.
HbAta+b HbAt~ HbAl Stable HbAla+b Stable HbAic Stable HbA~
n
Linear regression equation
r
P<
44 44 44 44 44 44
3' y y y y y
0.439 0.871 0.846 0.672 0.960 0.937
0.005 0.001 0.001 0.001 0.001 0.001
= = = = = =
0.76x 0.57x 0.45x 1.41x 0.66x 0.52x
as a solvent. Chromatograms were run at 280 nm and 254 nm. Tyrosine eluted under the same conditions as those for furosine determination was used as an internal standard. Furosine value was expressed as: furosine area at 280 nm tyrosine area at 280 nm
x
lO0 (%)
This was regarded as GHb-f value. Following the formation of furosine, 0.01 M phosphate buffer (pH 7.0) was incubated with 50 mM L-lysine and 50 mM glucose for 1 h at 100°C. Meanwhile, a portion of globin was incubated with NaBH4 at room temperature for 18 h, in which furosine component was unable to form.
+ + -
2.1 0.6 1.0 0.6 0.9 1.2
Results After acid hydrolysis of the sample formed by incubation of lysine and glucose, a single peak was eluted at the retention time of 4.1 min. The ratio of peak heights at 280 nm to 254 nm was 3.9:1 [1]. A globin sample also showed a peak with retention time of 4.1 min and the peak height ratio was also 3.9:1. The sample treated with NaBH4 before hydrolysis showed a remarkable decrease in this peak. Diabetic patients showed significantly higher GHb-f values than normal subjects, i.e., 4.6 -41.5% (mean + SD) versus 2.3 + 0.3% (P < 0.001). GHb-f values apparently correlated with HbA1 values (Table 1). In particular, the best positive correlation was found between GHb-f and stable HbAlc (r = 0.96, P < 0.001) (Fig. l).
(%) 1o,
n r V
=44 =0.960 =0.66x --0.90
p
/
.". ~ ,
Discussion
/"
GHb-f
,
Fig. 1. Correlation between G H b - f and stable HbA,¢ in diabetic patients. G H b - f represents the glycosylated hemoglobin determined by furosine measurement.
Concerning the binding of glucose and Hb, glycosylation has been reported to occur not only in fl chain N-terminal valine but in ct chain N-terminal or c~ or fl chain lysine residue. Bunn et al. [4] and Shapiro et al. [5] determined Hb by ion exchange column chromatography and each fraction of Hb by thiobarbituric acid (TBA) colorimetric assay and found that 5-hydroxymethyl furfural (5-HMF) was also produced from the HbAo fraction. They reported that 8-10% of the HbAo fraction was gly-
195
cosylated since the uptake of glucose was found when the isolated HbAo fraction was incubated with ~4C-glucose. Glycosylation of the HbAo fraction can be determined by the TBA method, affinity chromatography, etc. Affinity column chromatography in principle detects glucose bound to Hb as a result of a specific reaction to cis-diol groups. We also found a good correlation (r = 0.864, P < 0.001) between HbA1 values obtained by ion exchange column chromatography and by the TBA method [6]. However, TBA reacts with various substances containing aldehyde groups, and hence, the TBA reaction is not colorimetrically specific for 5HMF. It is expected that not only 5-HMF but various substances are generated from ketoamine by acid hydrolysis, and that those substances react with TBA [7]. Therefore, the TBA method possesses some problems [8]. The furosine assay method used in the present study was based on the principle that furosine is formed on acid hydrolysis from glucose which is bound to an amino group of the lysine residues [1]. GHb-f values were higher in diabetic patients than in normal subjects and there was a good correlation between HbA1 values and GHbf values. It appears that like HbAI, GHb-f can be used as an indicator of blood glucose control in diabetic patients. The furosine measurement needs a long time for hydrolysis but it is a new assay method by which glycosylation of the e-amino group of lysine can be specifically determined. This method is applicable to the determination of glycosylation of proteins, including food [3,9]. Glycosylation occurs widely in proteins in the living body, especially in patients with diabetic complications [10]. It has been reported that glycosylated proteins and polypeptides undergo changes in their biological activities [11]. This furosine assay method will be clinically useful in determining the glycosylation of not only Hb but also proteins.
References I Schleicher, E. and Wieland, O.H., Specific quantitation by HPLC of protein (lysine) bound glucose in human albumin and other glycosylated proteins, J. Clin. Chem. Clin. Biochem., 19: 81-87, 1981. 2 Nathan, D.M., Avezzano, E.S. and Palmer, J.L., A rapid chemical means for removing labile glycohemoglobin, Diabetes, 30: 700-701, 1981. 3 Oimomi, M., Hatanaka, H., Ishikawa, K., Kubota, S., Yosimura, Y. and Baba, S., Increased fructose-lysine of nail protein in diabetic patients, Klin. Wochenschr., 62: 477-478, 1984. 4 Bunn, H.F., Shapiro, R., McManus, M., Garrick, L., McDonald, M.J., Gallop, P.M. and Gabbay, K.H., Structural heterogeneity of human hemoglobin A due to nonenzymatic glycosylation, J. Biol. Chem., 254: 3892-3898, 1979. 5 Shapiro, R., McManus, M.J., Zalut, C. and Bunn, H.F., Site of nonenzymatic glycosylation of human hemoglobin A, J. Biol. Chem., 255: 3120-3127, 1980. 6 Kubota, S., Hatanaka, H., Ishikawa, K., Kawasaki, T., Takagi, K., Tanke, G., Yoshimura, Y., Oimomi, M. and Baba, S., Clinical application of HbA~ measured by affinity column method - - with special reference to diabetic nephropathy, Jpn. J. Clin. Pathol., 32: 555-557, 1984. 7 Nakayama, T., Hayase, F. and Kato, H., Formation of e(formyl-5-hydroxymethyl-pyrrol-l-yl)-L-norleucine in the Maillard reaction between D-glucose and L-lysine, Agric. Biol. Chem., 44: 1201-1202, 1980. 8 0 i m o m i , M., Kawasaki, T., Kubota, S., Yoshimura, Y., Baba, S. and Kato, H., Evaluation of colorimetric method using thiobarbituric acid for the determination of glycosylated hemoglobin, Acta Hematol. Jpn., 47: 868-872, 1984. 9 Vogt, B.W., Schleicher, E.D. and Wieland, O.H., e-Aminolysine-bound glucose in human tissue obtained at autopsy, Diabetes, 31: 1123-1127, 1982. 10 Kennedy, L. and Baynes, J.W., Non-enzymatic glycosylation and the chronic complications ofdiabetes; an overview, Diabetologia, 26: 93-98, 1984. II Oimomi, M., Hatanaka, H., Ishikawa, K., Kawasaki, T., Kubota, S., Yoshimura, T. and Baba, S., Effect of glycosylation on physiological and biological activities of substances, Arch. Gerontol. Geriatr., 3: 59-64, 1984.
Elsevier
193
DRC00030
A specific method of glycosylated hemoglobin using furosine measurement M u n e t a d a Oimomi 1, Shigeki Nishimoto, Shinichiro M a t s u m o t o , Hiroshi H a t a n a k a , K a z u o Ishikawa and Shigeaki Baba The Second Department of hzternal Medicine, Kobe University School of Medicine, Chuo-ku, Kobe, 650, Japan
(Received 22 February 1985,accepted 26 July 1985)
Key words: Glycosylatedhemoglobin;Fructose-lysine;Furosine; Diabetes mellitus
Summary In order to get an accurately specific determination of glycosylated hemoglobin (GHb-t), the furosine assay method, which can detect e-amino-lysine-bound glucose, was investigated. A good correlation was found between GHb-f values obtained by this method and HbA~ values by conventional ion exchange column chromatography. It was, therefore, considered that this technique would be a new useful method for determining glycosylated hemoglobin.
Introduction Recently it has become feasible to determine glycosylation of protein by measuring furosine derived from fructose-lysine, glucose bound to lysine residues of protein, by acid hydrolysis [1]. In the present study we investigated glycosylation of globin, a protein of hemoglobin (Hb).
Subjects and Methods Seven normal subjects and 44 diabetic patients were Correspondence to: Munetada Oimomi, M.D., The Second Department of Internal Medicine,Kobe UniversitySchool of Medicine, 5-2, 7-chome, Kusunoki-cho,Chuo-ku, Kobe, 650, Japan.
studied. Each blood sample collected in a heparinized syringe was determined for HbAI by high-performance liquid chromatography (HPLC, Auto HbAlc, Kyoto Daiichi Co.) using an ion exchange column method, and for glycosylated Hb by the furosine determination method (glycosylated Hb determined by furosine assay is referred to as GHb0. In determining stable HbA1, the method of Nathan et al. [2] was used. For the determination of GHb-f, globin was extracted with acid acetone from 500 #1 of blood and hydrolyzed with 2 ml of 6 N HCI for 30 h at 95°C [3]. After evaporation to dryness, 500/d of distilled water was added for reconstitution and the resultant solution was used as a sample. In the analysis of furosine, HPLC (Waters) and a TSK-gel column (ODS-120T, Toyo Soda Co.) were employed and 7 mM phosphate was used
0168-8227/85/$03.30 © 1985 ElsevierSciencePublishers B.V. (BiomedicalDivision)
194 TABLE 1 C O R R E L A T I O N BETWEEN G H b - f A N D HbA~ C O M P O N E N T S IN D I A B E T I C P A T I E N T S G H b - f represents the glycosylated hemoglobin determined by furosine measurement.
HbAta+b HbAt~ HbAl Stable HbAla+b Stable HbAic Stable HbA~
n
Linear regression equation
r
P<
44 44 44 44 44 44
3' y y y y y
0.439 0.871 0.846 0.672 0.960 0.937
0.005 0.001 0.001 0.001 0.001 0.001
= = = = = =
0.76x 0.57x 0.45x 1.41x 0.66x 0.52x
as a solvent. Chromatograms were run at 280 nm and 254 nm. Tyrosine eluted under the same conditions as those for furosine determination was used as an internal standard. Furosine value was expressed as: furosine area at 280 nm tyrosine area at 280 nm
x
lO0 (%)
This was regarded as GHb-f value. Following the formation of furosine, 0.01 M phosphate buffer (pH 7.0) was incubated with 50 mM L-lysine and 50 mM glucose for 1 h at 100°C. Meanwhile, a portion of globin was incubated with NaBH4 at room temperature for 18 h, in which furosine component was unable to form.
+ + -
2.1 0.6 1.0 0.6 0.9 1.2
Results After acid hydrolysis of the sample formed by incubation of lysine and glucose, a single peak was eluted at the retention time of 4.1 min. The ratio of peak heights at 280 nm to 254 nm was 3.9:1 [1]. A globin sample also showed a peak with retention time of 4.1 min and the peak height ratio was also 3.9:1. The sample treated with NaBH4 before hydrolysis showed a remarkable decrease in this peak. Diabetic patients showed significantly higher GHb-f values than normal subjects, i.e., 4.6 -41.5% (mean + SD) versus 2.3 + 0.3% (P < 0.001). GHb-f values apparently correlated with HbA1 values (Table 1). In particular, the best positive correlation was found between GHb-f and stable HbAlc (r = 0.96, P < 0.001) (Fig. l).
(%) 1o,
n r V
=44 =0.960 =0.66x --0.90
p
/
.". ~ ,
Discussion
/"
GHb-f
,
Fig. 1. Correlation between G H b - f and stable HbA,¢ in diabetic patients. G H b - f represents the glycosylated hemoglobin determined by furosine measurement.
Concerning the binding of glucose and Hb, glycosylation has been reported to occur not only in fl chain N-terminal valine but in ct chain N-terminal or c~ or fl chain lysine residue. Bunn et al. [4] and Shapiro et al. [5] determined Hb by ion exchange column chromatography and each fraction of Hb by thiobarbituric acid (TBA) colorimetric assay and found that 5-hydroxymethyl furfural (5-HMF) was also produced from the HbAo fraction. They reported that 8-10% of the HbAo fraction was gly-
195
cosylated since the uptake of glucose was found when the isolated HbAo fraction was incubated with ~4C-glucose. Glycosylation of the HbAo fraction can be determined by the TBA method, affinity chromatography, etc. Affinity column chromatography in principle detects glucose bound to Hb as a result of a specific reaction to cis-diol groups. We also found a good correlation (r = 0.864, P < 0.001) between HbA1 values obtained by ion exchange column chromatography and by the TBA method [6]. However, TBA reacts with various substances containing aldehyde groups, and hence, the TBA reaction is not colorimetrically specific for 5HMF. It is expected that not only 5-HMF but various substances are generated from ketoamine by acid hydrolysis, and that those substances react with TBA [7]. Therefore, the TBA method possesses some problems [8]. The furosine assay method used in the present study was based on the principle that furosine is formed on acid hydrolysis from glucose which is bound to an amino group of the lysine residues [1]. GHb-f values were higher in diabetic patients than in normal subjects and there was a good correlation between HbA1 values and GHbf values. It appears that like HbAI, GHb-f can be used as an indicator of blood glucose control in diabetic patients. The furosine measurement needs a long time for hydrolysis but it is a new assay method by which glycosylation of the e-amino group of lysine can be specifically determined. This method is applicable to the determination of glycosylation of proteins, including food [3,9]. Glycosylation occurs widely in proteins in the living body, especially in patients with diabetic complications [10]. It has been reported that glycosylated proteins and polypeptides undergo changes in their biological activities [11]. This furosine assay method will be clinically useful in determining the glycosylation of not only Hb but also proteins.
References I Schleicher, E. and Wieland, O.H., Specific quantitation by HPLC of protein (lysine) bound glucose in human albumin and other glycosylated proteins, J. Clin. Chem. Clin. Biochem., 19: 81-87, 1981. 2 Nathan, D.M., Avezzano, E.S. and Palmer, J.L., A rapid chemical means for removing labile glycohemoglobin, Diabetes, 30: 700-701, 1981. 3 Oimomi, M., Hatanaka, H., Ishikawa, K., Kubota, S., Yosimura, Y. and Baba, S., Increased fructose-lysine of nail protein in diabetic patients, Klin. Wochenschr., 62: 477-478, 1984. 4 Bunn, H.F., Shapiro, R., McManus, M., Garrick, L., McDonald, M.J., Gallop, P.M. and Gabbay, K.H., Structural heterogeneity of human hemoglobin A due to nonenzymatic glycosylation, J. Biol. Chem., 254: 3892-3898, 1979. 5 Shapiro, R., McManus, M.J., Zalut, C. and Bunn, H.F., Site of nonenzymatic glycosylation of human hemoglobin A, J. Biol. Chem., 255: 3120-3127, 1980. 6 Kubota, S., Hatanaka, H., Ishikawa, K., Kawasaki, T., Takagi, K., Tanke, G., Yoshimura, Y., Oimomi, M. and Baba, S., Clinical application of HbA~ measured by affinity column method - - with special reference to diabetic nephropathy, Jpn. J. Clin. Pathol., 32: 555-557, 1984. 7 Nakayama, T., Hayase, F. and Kato, H., Formation of e(formyl-5-hydroxymethyl-pyrrol-l-yl)-L-norleucine in the Maillard reaction between D-glucose and L-lysine, Agric. Biol. Chem., 44: 1201-1202, 1980. 8 0 i m o m i , M., Kawasaki, T., Kubota, S., Yoshimura, Y., Baba, S. and Kato, H., Evaluation of colorimetric method using thiobarbituric acid for the determination of glycosylated hemoglobin, Acta Hematol. Jpn., 47: 868-872, 1984. 9 Vogt, B.W., Schleicher, E.D. and Wieland, O.H., e-Aminolysine-bound glucose in human tissue obtained at autopsy, Diabetes, 31: 1123-1127, 1982. 10 Kennedy, L. and Baynes, J.W., Non-enzymatic glycosylation and the chronic complications ofdiabetes; an overview, Diabetologia, 26: 93-98, 1984. II Oimomi, M., Hatanaka, H., Ishikawa, K., Kawasaki, T., Kubota, S., Yoshimura, T. and Baba, S., Effect of glycosylation on physiological and biological activities of substances, Arch. Gerontol. Geriatr., 3: 59-64, 1984.