Clinica Chimica Acra. 212 (1992) 133-139
133
0 1992 Elsevier Science Publishers B.V. All rights
CCA
reserved.0009-8981/92/$05.00
05422
Short Communication
A calorimetric microassay for glycated collagen based on the thiobarbituric acid method Nessar Ahmed, Nageena S. Malik and Anna J. Furth Biophysics Group, Oxford Research Unit, The Open University, Boars Hill, Oxford OX1 SHR (UK)
(Received 10 February 1992; revision received 10 August 1992; accepted 9 September 1992)
Key words: Bovine serum albumin; Human serum albumin; Thiobarbituric acid; Hydroxymethyl furfural;
Glycation
Introduction Glycation is a spontaneous reaction between reducing sugars in the acyclic form and protein amino groups. The first product is a labile aldimine or Schiff base. Some of this SB will rearrange to form a more stable ketoamine or Amadori product. Measurement of glycated proteins such as haemoglobin [l] and albumin [2] are of interest in clinical laboratories in the assessment of blood glucose control in diabetics. Glycation of long-lived proteins such as collagen has been implicated in the long term complications of diabetes [3] and measurement of such proteins is of interest in research laboratories. The thiobarbituric acid (TBA) assay is one of the most widely used calorimetric methods for measurement of protein glycosylation. The glycated protein is subjected to mild acid hydrolysis so that the Amadori product form releases hydroxymethylfurfural (HMF). This HMF is then reacted with TBA to produce a chromophore the concentration of which can be measured photometrically. This method has the advantage that the Schiff bases do not interfere and neither do collagen crosslinks [4] and so the method has been used to measure glycated albumin [5], haemoglobin [6] and collagen [7,8]. Free sugars do interfere in the assay but can be removed readily by dialysis. Schleicher et al. have suggested that enzymatically bound sugars interfere in the TBA method [9]. Correspondence lo: Nageena S. Malik, Biophysics Group, Oxford Research Unit, The Open University, Boars Hill, Oxford OX1 5HR, UK.
134
The major disadvantages of the TBA method are that it is cumbersome, unable to provide absolute concentrations of Amadori product (hence protein glycosylation) but does allow ‘relative’ amounts of protein glycosylation to be compared, it is also non-stoichiometric and gives poor yields of HMF. The autoclave version of the TBA method introduced by Parker et al [6] has the advantage in that it only requires 1 h of hydrolysis compared to earlier versions which needed between 4-8 h [5,10]. For these reasons it has been superseded by other methods for measuring glycated haemoglobin and albumin in routine laboratories. Despite these disadvantages, it is still a simple and inexpensive method for measuring glycated collagen. We have further modified the method of Parker et al. and adapted it for use with a microplate reader. This modified method has an improved yield of HMF and increased sensitivity requiring only 1 mg of protein. Using in vitro glucated collagen as a model glycoprotein, we have estimated that the total interference by enzymatically bound sugar is < 5%. Our method was used to estimate in vivo glycation of human serum albumin (HSA) and compared well with published values obtained by other assays. Furthermore an excellent correlation coefficient was obtained for our assay and the stoichiometric periodate method. With the microplate reader, up to 94 samples can be read in 40 s. Materials and Methods Chemicals and equipment
Bovine tendon collagen type I, human serum albumin (HSA), collagenase (EC 3.4.24.8), papain (EC 3.4.22.2) and hydroxymethylfurfural were obtained from Sigma Chemicals (Poole, Dorset, UK). Bovine serum albumin (fraction V) was obtained from BDH Chemicals (Atherstone, Warwick, UK). Absorbance was read on a Bio-tek automated microplate reader, model EL31 1 using a 450 nm filter (Bio-tek Instruments, Luminar Technology, Swanmore, Hampshire, UK). In vitro glycation
Collagen (40 &pl) or BSA (10 &PI) was incubated in 0.5 M glucose for up to 20 days at 37°C in 0.05 M sodium phosphate buffer, pH 7.4, containing 3 mM sodium azide. The samples were dialyzed exhaustively against water to remove unbound glucose and stored frozen. Borohydride reduction of collagen
Dialyzed glycated collagen was stirred overnight at 37°C in 55 mM sodium borohydride in 1 mM NaOH, pH 7.4. The reduction was terminated by reducing the pH to 3.0 using concentrated acetic acid and the samples dialysed exhaustively against water to remove any unreacted borohydride. Protein concentration
The collagen concentration
of glycated samples was estimated using the hydroxy-
135
proline method [ 111after pretreatment with collagenase. Albumin concentration was determined using the Pierce BCA assay reagent based on the method of Smith et al H21. Collagen digestion
The dialyzed glycated collagen was centrifuged at 9,000 x g for 10 min and the precipitate resuspended in 0.05 M phosphate buffer, pH 7.4, containing 200 IU collagenase and incubated at 37°C for 48 h. Chloroform and toluene (5 ~1 each) were added to prevent bacterial growth. After centrifugation at 9,~ x g for 10 min, the supernatant was collected and stored frozen. TBA microassay procedure
To 100 ~1 of protein solution, fructose or HMF standard in bijoux bottles was added 100 ~1 of 0.5 M oxalic acid. The bottles were tightly sealed and incubated for 60 min in a pressure cooker at 124°C and 124 kPa pressure. The samples were allowed to cool for 15 min at room temperature and then transferred to eppendorf tubes. To these samples 100 ~1 of 2.45 M trichloroacetic acid (TCA) was added. After centrifugation at 9,000 x g for 10 min, 200-~1 portions of the supernatant were transferred to microplate wells, To these, 67 ~1 of 0.05 M TBA (which had been carefully adjusted to pH 6.0 with NaOH) was added to each well and mixed thoroughly. Microplates were incubated at 37°C for 30 min to allow the chromophore to develop and then allowed to cool to room temperature for 15 min. Reagent blanks (200 ~1 of water and 67 ~1 of TBA) were incubated at the same time. Absorbance measurements were made at 450 nm using the microplate autoreader. HMF loss during hydrolysis
Two sets of HMF standards were prepared and treated exactly as in the standard procedure except that the boiling step was omitted in one of the sets. By comparing the intensities of chromophore produced, the percentage loss of HMF during boiling was determined. Interference by glycoproteins
The chromophore yield from glycated collagen before and after borohydride reduction was used to estimate the degree of interference by enzymatically-bound sugars and any other substances. Correlation between TBA and periodate assays
The extent of glycation of 22 BSA samples incubated in glucose for different periods of time was measured by both the TBA microassay and our previously described periodate microassay [13]. These values were used to determine the correlation coefficient (r) between the two methods.
136
In vivo glycation measurements of human cornea1 and scleral collagen
Collagen was extracted from human corneas and scleras (approx. 5- 10 mg tissue) taking care to wash out the proteoglycans which are a major source of 6-carbon sugars [14]. The extracted samples were subjected to borohydride reduction, as described elsewhere and following this were digested with the enzyme papain (0.5 &pl citrate buffer, pH 6) at 55°C for 24 h. Results and Discussion Fructose standards were used to prepare a calibration graph for the TBA method, since fructose resembles the Amadori product form of glycated protein and releases HMF upon mild acid hydrolysis. Figure 1 shows that absorbance is linear in the range O-50 nmol fructose. One of the major criticisms of the TBA method is that it is non-stoichiometric since the net amount of HMF available for chromophore formation depends on the balance between production and loss during the heating process. For example, Parker et al. have reported 30% loss of HMF after 1 h of heating in an autoclave. Furthermore, production of HMF depends on protein concentration and is higher with less protein [15]. Since our assay requires less protein than earlier versions, a
0.6-
0.4-
E
C s
133
0 1992 Elsevier Science Publishers B.V. All rights
CCA
reserved.0009-8981/92/$05.00
05422
Short Communication
A calorimetric microassay for glycated collagen based on the thiobarbituric acid method Nessar Ahmed, Nageena S. Malik and Anna J. Furth Biophysics Group, Oxford Research Unit, The Open University, Boars Hill, Oxford OX1 SHR (UK)
(Received 10 February 1992; revision received 10 August 1992; accepted 9 September 1992)
Key words: Bovine serum albumin; Human serum albumin; Thiobarbituric acid; Hydroxymethyl furfural;
Glycation
Introduction Glycation is a spontaneous reaction between reducing sugars in the acyclic form and protein amino groups. The first product is a labile aldimine or Schiff base. Some of this SB will rearrange to form a more stable ketoamine or Amadori product. Measurement of glycated proteins such as haemoglobin [l] and albumin [2] are of interest in clinical laboratories in the assessment of blood glucose control in diabetics. Glycation of long-lived proteins such as collagen has been implicated in the long term complications of diabetes [3] and measurement of such proteins is of interest in research laboratories. The thiobarbituric acid (TBA) assay is one of the most widely used calorimetric methods for measurement of protein glycosylation. The glycated protein is subjected to mild acid hydrolysis so that the Amadori product form releases hydroxymethylfurfural (HMF). This HMF is then reacted with TBA to produce a chromophore the concentration of which can be measured photometrically. This method has the advantage that the Schiff bases do not interfere and neither do collagen crosslinks [4] and so the method has been used to measure glycated albumin [5], haemoglobin [6] and collagen [7,8]. Free sugars do interfere in the assay but can be removed readily by dialysis. Schleicher et al. have suggested that enzymatically bound sugars interfere in the TBA method [9]. Correspondence lo: Nageena S. Malik, Biophysics Group, Oxford Research Unit, The Open University, Boars Hill, Oxford OX1 5HR, UK.
134
The major disadvantages of the TBA method are that it is cumbersome, unable to provide absolute concentrations of Amadori product (hence protein glycosylation) but does allow ‘relative’ amounts of protein glycosylation to be compared, it is also non-stoichiometric and gives poor yields of HMF. The autoclave version of the TBA method introduced by Parker et al [6] has the advantage in that it only requires 1 h of hydrolysis compared to earlier versions which needed between 4-8 h [5,10]. For these reasons it has been superseded by other methods for measuring glycated haemoglobin and albumin in routine laboratories. Despite these disadvantages, it is still a simple and inexpensive method for measuring glycated collagen. We have further modified the method of Parker et al. and adapted it for use with a microplate reader. This modified method has an improved yield of HMF and increased sensitivity requiring only 1 mg of protein. Using in vitro glucated collagen as a model glycoprotein, we have estimated that the total interference by enzymatically bound sugar is < 5%. Our method was used to estimate in vivo glycation of human serum albumin (HSA) and compared well with published values obtained by other assays. Furthermore an excellent correlation coefficient was obtained for our assay and the stoichiometric periodate method. With the microplate reader, up to 94 samples can be read in 40 s. Materials and Methods Chemicals and equipment
Bovine tendon collagen type I, human serum albumin (HSA), collagenase (EC 3.4.24.8), papain (EC 3.4.22.2) and hydroxymethylfurfural were obtained from Sigma Chemicals (Poole, Dorset, UK). Bovine serum albumin (fraction V) was obtained from BDH Chemicals (Atherstone, Warwick, UK). Absorbance was read on a Bio-tek automated microplate reader, model EL31 1 using a 450 nm filter (Bio-tek Instruments, Luminar Technology, Swanmore, Hampshire, UK). In vitro glycation
Collagen (40 &pl) or BSA (10 &PI) was incubated in 0.5 M glucose for up to 20 days at 37°C in 0.05 M sodium phosphate buffer, pH 7.4, containing 3 mM sodium azide. The samples were dialyzed exhaustively against water to remove unbound glucose and stored frozen. Borohydride reduction of collagen
Dialyzed glycated collagen was stirred overnight at 37°C in 55 mM sodium borohydride in 1 mM NaOH, pH 7.4. The reduction was terminated by reducing the pH to 3.0 using concentrated acetic acid and the samples dialysed exhaustively against water to remove any unreacted borohydride. Protein concentration
The collagen concentration
of glycated samples was estimated using the hydroxy-
135
proline method [ 111after pretreatment with collagenase. Albumin concentration was determined using the Pierce BCA assay reagent based on the method of Smith et al H21. Collagen digestion
The dialyzed glycated collagen was centrifuged at 9,000 x g for 10 min and the precipitate resuspended in 0.05 M phosphate buffer, pH 7.4, containing 200 IU collagenase and incubated at 37°C for 48 h. Chloroform and toluene (5 ~1 each) were added to prevent bacterial growth. After centrifugation at 9,~ x g for 10 min, the supernatant was collected and stored frozen. TBA microassay procedure
To 100 ~1 of protein solution, fructose or HMF standard in bijoux bottles was added 100 ~1 of 0.5 M oxalic acid. The bottles were tightly sealed and incubated for 60 min in a pressure cooker at 124°C and 124 kPa pressure. The samples were allowed to cool for 15 min at room temperature and then transferred to eppendorf tubes. To these samples 100 ~1 of 2.45 M trichloroacetic acid (TCA) was added. After centrifugation at 9,000 x g for 10 min, 200-~1 portions of the supernatant were transferred to microplate wells, To these, 67 ~1 of 0.05 M TBA (which had been carefully adjusted to pH 6.0 with NaOH) was added to each well and mixed thoroughly. Microplates were incubated at 37°C for 30 min to allow the chromophore to develop and then allowed to cool to room temperature for 15 min. Reagent blanks (200 ~1 of water and 67 ~1 of TBA) were incubated at the same time. Absorbance measurements were made at 450 nm using the microplate autoreader. HMF loss during hydrolysis
Two sets of HMF standards were prepared and treated exactly as in the standard procedure except that the boiling step was omitted in one of the sets. By comparing the intensities of chromophore produced, the percentage loss of HMF during boiling was determined. Interference by glycoproteins
The chromophore yield from glycated collagen before and after borohydride reduction was used to estimate the degree of interference by enzymatically-bound sugars and any other substances. Correlation between TBA and periodate assays
The extent of glycation of 22 BSA samples incubated in glucose for different periods of time was measured by both the TBA microassay and our previously described periodate microassay [13]. These values were used to determine the correlation coefficient (r) between the two methods.
136
In vivo glycation measurements of human cornea1 and scleral collagen
Collagen was extracted from human corneas and scleras (approx. 5- 10 mg tissue) taking care to wash out the proteoglycans which are a major source of 6-carbon sugars [14]. The extracted samples were subjected to borohydride reduction, as described elsewhere and following this were digested with the enzyme papain (0.5 &pl citrate buffer, pH 6) at 55°C for 24 h. Results and Discussion Fructose standards were used to prepare a calibration graph for the TBA method, since fructose resembles the Amadori product form of glycated protein and releases HMF upon mild acid hydrolysis. Figure 1 shows that absorbance is linear in the range O-50 nmol fructose. One of the major criticisms of the TBA method is that it is non-stoichiometric since the net amount of HMF available for chromophore formation depends on the balance between production and loss during the heating process. For example, Parker et al. have reported 30% loss of HMF after 1 h of heating in an autoclave. Furthermore, production of HMF depends on protein concentration and is higher with less protein [15]. Since our assay requires less protein than earlier versions, a
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