Clinica Chimica Acta, 212 (1992) 3-15 @ 1992 Elsevier Science Publishers B.V. All rights reserved. 0009-8981/92/%05.00

CCA 05409

Measurement of glycated albumin by the nitroblue tetrazolium calorimetric method Shinichi Mashiba”, Kazuo Uchida”, Shoji Okuda” and Shinobu Tomitab “Kyoto Medical Science Laboratory Incorporation, Furukawa-cho, 328 Hazukashi Fushimi-ku. Kyoto 612 and ‘Kyoto Hakuaikai Hospital, I Kamigamo, Kita-ku, Kyoto 603 (Japan)

(Received 29 February 1992; revision received 21 August 1992; accepted 31 August 1992)

Key words: Nitroblue tetrazolium; Albumin fructosamine; Glycated albumin; Polyethylene glycol

Summary A method has been developed for the measurement of glycated albumin (albuminfructosamine) by the nitroblue tetrazolium (NBT) calorimetric method. In this method, polyethylene glycol was added to the serum and then the mixture was centrifuged to separate globulin proteins from albumin proteins. This made it possible to measure the glycated albumin in the supernatant by the NBT calorimetric method, without the interference of globulin proteins. This measurement method correlated with the measurement of glycated albumin using boronate affinity chromatography with an r value of 0.942 (P < 0.001). Our method using polyethylene glycol permits easy measurement of albumin fructosamine and is therefore useful as an index of diabetic control and for diabetic screening.

Introduction Assays for glycated hemoglobin and fructosamine are well-known indices of past blood-sugar levels in diabetic patients. Glycated hemoglobin reflects the average blood-sugar value over a period of two to three months prior to the time of measurement since the half-life of hemoglobin is relatively long [l]. The measurement of glycated albumin by the nitroblue tetrazolium (NBT) calorimetric method was Correspondence to: Shinichi Mashiba, Kyoto Medical Science Laboratory Hazukashi Fushimi-ku, Kyoto 612, Japan.

Inc., Furukawacho

328,

4

developed by Johnson et al. [2]. The assay is based on the principle of NBT reduction by the ketoamine moiety of glycated protein in an alkaline solution [2]. It is felt that the fructosamine assay mainly reflects the glycation of albumin. The assay thus estimates the blood-sugar value for l-2 weeks prior to the measurement because the half-life of albumin is approx. 17 days [3]. However, it is known that many proteins of different half-lives, including apolipoproteins and Ig-A, are glycated non-enzymatically [4,5]. Changes occur in globulin proteins in patients with diabetes. When globulin proteins are glycated, the fructosamine measurement is affected by their reducing activity. For example, a patient with a normal albumin concentration and an elevated blood globulin concentration, will have an artifactually high fructosamine concentration [5]. The fructosamine value in a patient correlates with albumin concentration [3]. Thus, there are several problems associated with the use of the fructosamine assay as an index of glycemic control. However, no major efforts have been undertaken to resolve the problems of the fructosamine assay. Methods for the specific measurement of glycated albumin include boronate aflinity chromatography [6], high-performance liquid chromatography (HPLC) [7], radioimmunoassay [8] and aft?gel blue [9]. These methods have shortcomings in terms of simplicity and measurement accuracy. We have developed an albuminfructosamine assay that overcomes these problems. Serum albumin is separated from serum globulin by means of polyethylene glycol. Then, the NBT reducing activity of the ketoamine of the serum albumin is determined in an alkali solution. Materials and Methods Subjects Patients with diabetes, paraproteinemia, hypoproteinemia and hypo-albuminemia and normal control subjects who underwent a physical examination were studied. Fractionation of serum albumin with polyethylene glycol Five hundred microliters of serum was added to 50 mg of polyethylene glycol (PEG) 6000 (Nakarai Tesque, Japan) and stirred. The mixture was centrifuged at 2000 x g for 5 min. Albumin was located in the supernatant and globulin proteins were precipitated. At the polyethylene glycol concentration used, globulins were efficiently removed from serum containing a wide range of protein concentrations without greatly reducing the serum albumin concentration. The polyethylene glycol was prepared as a powder so that the added serum would dissolve rapidly with no effect on the reaction system as compared with other deproteinization agents. Assay of albumin fructosamine The concentration of albumin fructosamine in the supernatant was measured by the NBT calorimetric method using an Hitachi 7 150 discrete analyzer (Hitachi Ltd., Japan). Fructosamine from human serum was calibrated with glycated polylysine

5

(F. Hoffmann-La Roche, Basle, Switzerland) and used as the reference standard. Commercial kits (F. Hoffmann-La Roche, Basle, Switzerland) were used for the reagents. The composition of the reagents and the conditions of analysis are given in Table I. Assay of fructosamine

Fructosamine was measured in unseparated samples. The compositions of reagents and analytical conditions employed are shown in Table I for the albuminfructosamine assay. Measurement of glycated albumin using boronate affinity gel

Twenty-five microliters of serum was applied to an affinity gel column to which m-aminophenylboronic acid was fixed. Non-glycated proteins were eluted with buffer 1 (0.25 mol/l ammonium acetate buffer solution, pH 8.5, 0.05 mol/l MgClz). The glycated proteins were eluted with buffer 2 (0.1 mol/l Tris-buffer, pH 8.5, 0.2 mol/l D-sorbitol). Albumin concentrations in the fractions were determined by immunonephelometry. The procedure for immuno-nephelometry was as follows. The reagent was prepared by diluting antihuman albumin antibody (DAKOPATTS, Denmark) lOOfold with a phosphate buffer solution (10 nmol/l Na2HP04, 5 mmol/I NaH2P04, 150 mmolil NaCl, pH 7.2) containing 5 g/l PEG6000. The standard was prepared with the amount of human albumin (Sigma, A1653) adjusted to 25-200 &ml. Each fraction and standard was measured by means of an Hitachi 705 discrete analyzer (Hitachi Ltd. Japan). The measurement conditions for the Hitachi 705 were: sample volume 20 ~1, reagent volume 400 ~1, wavelength 340/700 nm, reaction time 10 min and reaction temperature 37°C. Albumin concentration of each fraction was calculated by comparing the optical absorption of each fraction with those of the standards.

TABLEr Reagents and instrument settings for Hitachi 7 150 analyzer Reagent composition

Nitroblue tetrazolium Sodium carbonate buffer Detergent Uricase

0.48 mmolil 200 mmol/l, pH 10.3 22 g/l >2.5 kunitsfl

Assay settings

Standard cont. Sample vol. Reagent vol. Wavelength Measurement interval Temperature

479 gmol/l 14 pl 250 pl 546/700 nm 9-10 min 37°C

Results Removal of globulin proteins with polyethylene glycol

The cellulose acetate membrane ele~trophoretic method (Helena Laboratories) was used on serum sampies obtained from three patients with different globulin protein compositions. Celfulose acetate membrane el~trophoreti~ patterns are shown in Fig. 1. With poIyethylene glycol precipitation, the major component of the supernatant was albumin, indicating that oz2-,& and ~-~obulin were removed. AIb~in ~o~~ent~tions before and after PEG processing were compared for 100 sampies of sera obtained from patients with different albumin concentrations, and as shown in Fig. 2, albumin concentrations did not change greatly. Cellulose acetate membrane eIectrophoresis was used for serum samples from many patients with different lipoprotein compositions. Pre-0 and P-lipoprotein were completely removed from the supernatant which was separated using PEG. Removal of NBT color reducing activity of glycated globulin proteins with PEG

Serum samples with high fructosamine vdues were used. We adopted the method of Kobayashi et al. IlO]. Two pieces of cellulose acetate membrane (TITAN III, Heiena ~~ratories) were used for each test. Three ~croliters of each sample was applied to one of the membranes and ele~trophoresis was induced (6 mA for 20 min). The other cellulose acetate membrane was soaked in 0.1 M/l carbonate buffer (pH 10.3) containing 5 mM NBT (WAKO Junyaku, Japan). These two membranes were laid one atop the other and allowed to react at 37’C overnight. Then, the NBT reducing activity of the glycated protein fractions was measured. For unseparated serum, both albumin and ~-globulin exhibited NBT reducing activity. However, following PEG separation, NBT reducing activity was observed only for albumin. within-rub and between-rub reproducibility of the alb~~~~ fr~ctosa~i~e assay and the frucYosa~~ne assay

The within-run reproducibility of the albumin fructosamine assay and the fructosamine assay was examined using two kinds of serum. As shown in Table II, both assays demonstrated good reproducibility. With regard to daily errors, both assays demonstrated good reprodu~ibiiity, as shown in Table III. Effects of reducing s#bsta~ces

The effects of reducing substances are shown in Fig. 3. Glucose, uric acid, reduced-form glutathione, ascorbic acid, intralipid, free bilirubin, conjugated bili~bin and hemo~obin were added to pooled serum from normal subjects. The effects of these reproducing substances were compared for the fructosamine assay and the present albumin-f~~tosa~ne method. Intralipid had no elect on either assay. However, glucose, uric acid, reduced-form glutathione, free bilirubin and con-

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Fig. 1. Cellulose acetate membrane electrophoresis of the initial serum sample before and after PEG application. Cellulose acetate membrane electrophoretic patterns of serum samples from three patients. Percentage, total protein, albumin, fructosamine and albumin-fructosamine values are shown. Total protein was measured by the biuret method and albumin by the bromocresol purple method using an Hitachi 736 discrete analyzer (Hitachi Ltd., Japan).

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Fig. 2. Measurement of serum albumin before and after PEG segregation. Serum albumin was measured by the bromocresol purple method, using an Hitachi 736 discrete analyzer (Hitachi Ltd. Japan).

jugated bilirubin increased the readings for both assays, while ascorbic acid lowered the readings for both assays. At a hemoglobin concentration of 1000 mg/dl, the fructosamine assay read 45% low whereas the present method read 24% low. Precipitation of hemoglobin with PEG decreased the negative error. The removal of globulin proteins from pooled serum explained the shift to lower values by the present method.

TABLE II ‘Within-run’ reproducibility of the fructosamine assay and the albumin fructosamine assay n = 30

Mean (pmoV1) S.D. (amol/I) cv (%)

Albumin fructosamine

Fructosamine Sample 1

Sample 2

Sample 1

Sample 2

240.5 1.5 0.6

421.0 3.8 0.9

186.5 1.4 0.8

334.9 3.9 1.1

9 TABLE III ‘Between-run’ reproducibility of the fructosamine assay and the albumin fructosamine assay n = 10

Mean bmolfl) S.D. (pmol/l) cv (%)

Albumin fructosamine

l+-uctosamine Sample 1

Sample 2

Sample 1

Sample 2

239.1 3.4 1.4

423.8 4.3 1.0

185.0 2.2. 1.2

339.5 4.7 1.4

Correlation among glycated albumin measurement by boronate affinity gel, fructosamine and albumin-fructosamine assays

Measurements were performed on serum samples from 184 patients, including 4 patients with cirrhotic liver and 34 patients with multiple myeloma. Figure 4 shows the relationship between glycated albumin measurements by boronate affinity gel, fructosamine and albumin-fructosamine (the present method). Compared with fructosamine measurements, the regression line using the present method was nearly straight, passed through the origin and more points converged on the regression line. The correlation coefficient improved from a value of r = 0.916 to r = 0.942. Significance tests were conducted for the correlation coefficients of r = 0.916 and r = 0.942 and a significant difference was found for P < 0.05. Normal range

Comparison of normal values for fructosamine measurements and albumin fructosamine measurements was carried out, using sera obtained from 300 healthy subjects (Fig. 5). Normal values for fructosamine measurements were between 226 and 291 (rmol/l) and those for albumin fructosamine were between 160 and 222 (pmol/l). Compared with fructosamine measurements, the normal value shifted to the lower side because the current method suppressed the contribution of glycated proteins, other than albumin, to the measurements. Serum albumin fructosamine values in diabetic patients

Serum albumin fructosamine measurements were carried out on insulin-dependent diabetes mellitus (IDDM) patients whose mean blood glucose value was 181.1 i 92.5 mg/dl (48 cases) and non-insulin-dependent diabetes mellitus (NIDDM) patients whose blood glucose value was 175.7 f 73.2 mg/dl (29 cases). The results were as follows: The albumin fructosamine value for IDDM patients was 424.6 * 83.6 pmol/l and that for NIDDM patients was 346.5 * 61.6 pmol/l. These values were high compared with those obtained from normal subjects (Fig. 6).

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Fig. 3. Effects of reducing

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Fig. 4. Correlation among measurements of glycated albmnin by boronate affinity gel, fructosamine and with albumin-fructosamine methods.

12

I;ructosamine

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290

( Ltml/l)

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Albumin Fructosamine 40

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290 ( Pmol/l)

Fig. 5. Normal values from the fructosamine measurements and albumin fructosamine measurements.

Discussion Albumin, the major serum protein, has been considered most likely to undergo glycation. Fructosamine has been thought to reflect the reducing activity of glycated albumin [ 11,121. However, it has been demonstrated that many proteins in the

13

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IDDM (N=18) Fig. 6. Serum albumin fructosamine

NIDDM (N=29)

values from 48 IDDM cases and 29 NIDDM

cases,

globulin fraction undergo non-enzymatic glycation. Glycated proteins such as fllipoprotein [ 13,141 and monoclonal para proteins (particularly, Ig-A K) [ 151 and others have great NBT reducing activity. It has been suggested that these proteins have an effect on the fructosamine measurement value. We have developed a simple and easy method for measurement of albumin-fructosamine using the NBT colorimetric method, in which the effect of glycated globulin proteins is eliminated through PEG separation. This method eliminates the NBT reducing activity and the glycated proteins in the globulin protein fraction and leaves intact the NBT reducing activity of glycated albumin. Accordingly, as shown by the comparison of the glycated albumin measurement method using boronate affinity gels, the fructosamine measurement

14

method and the current albumin fructosamine measurement method, the results obtained with the current method had good correlations with the glycated albumin values obtained by the boronate affinity gel method. Addition of hemoglobin as a reducing substance resulted in smaller negative errors under the present method than in the fructosamine method. PEG precipitated hemoglobin as well as globulin proteins. This suggests that proteins with NBT reducing activity, other than glycated proteins, can be removed by PEG separation. The effects of bilirubin and ascorbic acid on the present method may be reduced with the addition of bilirubin oxidase and ascorbate oxidase. In fact, uricase and surfactants have recently been incorporated into commercial fructosamine reagents. However, it is difficult to completely eliminate the reducing activity of small molecules by methods based on the NBT calorimetric method. Glycated albumin represents non-enzymatic binding of glucose to a lysine residue containing an E amino group. The boronate affinity gel technique is based on selective binding of glycated albumin to an m-aminophenyl-boronic acid-fixed column. It has been reported that the albumin molecule has 59 lysine residues and that four surface lysine residues can be modified [ 161. In experiments using [ “C]glucose, it was calculated that 4.7 molecules of glucose bind to an albumin molecule [17]. The glycated albumin assay based on boronate affinity gel (including the HPLC method), counts a single glycated lysine residue as well as multiple glycated residues as one glycated albumin molecule [18]. The present method, however, measures the full extent of glycation because it accurately determines the number of ketoamines which reduce NBT. Nevertheless, evaluation of the current results showed no clear distinction between the two methods. The glycated albumin assay is complicated and diBicult to perform. The HPLC method is not suitable for screening although it is easy to perform. Walker et al. [9] used afti-gel blue for separation of albumin in the development of a method for glycated albumin measurements utilizing the NBT calorimetric technique. However, Walker’s afti-gel blue assay procedure is as complex as the boronate affinity gel assaying procedure and is not suitable for routine measurements and screening. Our method using PEG permits easy measurement of albumin-fructosamine. The measurement is useful as an index of diabetic control and for diabetic screening. It can be applied to various types of automatic analyzers with simplicity and excellent measurement accuracy. References 1 Dunn PJ, Cole RA, Soeldner JS et al. Temporal relationships of glycosylated haemoglobin concentrations to glucose control in diabetics. Diabetologia 1979;17:213-220. 2 Johnson R, Metcalf PA, Baker JR. Fructosamine: a new approach to the estimation of serum glycosylation. An index of diabetic control. Chn Chim Acta 1982;127:87-95. 3 Kurahachi H, Moridera K et al. Clinical usefulness of serum fructosamine levels in diabetics. J Jpn Diab Sot 1987;30:987-994. 4 Steinbrecher UP, W&turn JL. Glucosylation of low density lipoprotein to an extent comparable to that seen in diabetes slows their catabolism. Diabetes 1984;33:130-134. 5 Rodriguez S et al. Effects of various serum proteins on quantification of fructosamine. Clin Chem 1989;35:134-138.

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8 9 10 11 12 13 14 15 16 17 18

Yatscoff RW, Tevaarwerk GJM, MacDonald JC. Quantification of nonenzymatically glycated albumin and total serum protein by afftnity chromatography. Clin Chem 1984;30:446-449. Abe F, Yano M, Hirota M et al. High-performance liquid chromatographic assay of serum glycated albumin, with special reference to a comparison of its assay conditions with those of fructosamine. J Jpn Diab Sot 1989;32:183-188. Taneda S, Nakayama H et al. Radioimmunoassay of nonenzymatically glucosylated albumin. J Jpn Diab Sot 1986;29:581-590. Walker SW, Howie AF, Smith AF. The measurement of glycosylated albumin by reduction of alkaline nitro-blue tetrazolium. Clin Chim Acta 1986;156:197-206. Kobayashi K, Ogasahara N, Sakoguchi T et al. Serum glycated &lipoprotein determined by agarose-gel electrophoresis with nitroblue tetrazolium coloration. Clin Chem 1989;35:177-178. Romey CH, Pascual C. Nitroblue tetrazolium staining of serum fructosamine on agarose gel electrophoretograms (Letter), Clin Chem 1987;33:1949. Johnson RN, Baker JR. The alkaline reducing activity of glycated serum proteins and its relevance to diabetes mellitus. Clin Chem 1986;32:368-370. Kobayashi K, Ogasahara N, Matsuoka A. Glycation of human serum albumin in long-term incubation with low and high concentrations of glucose, Chem Pharm Bull 1991;39:1080-1081. Kobayashi K, Ogasahara N, Matsuoka A, et al. Correlation between glycated lipoproteins and fructosamine level in serum. Chem Pharm Bull 1991;39:2149-2151. Sivas A, Unal T, Oz H. Effect of IgA light chain on concentration of fructosamine in serum (Letter). Clin Chem 1990;36:1386-1387. Dayhoff MO. Atlas of protein sequence and structure. National Biomedical Foundation, Washington D. C., Vol. 5, Suppl. 2, 1976. Naka E. Measurement of fructosamine. Jpn Mod Med Lab 1989;17:1055-1060. Ikegami H, Ogihara T. Glycated serum protein (GSP), glycated albumin (GA) and fructosamine. Jpn J Clin Med 1990;48:395-400.

Measurement of glycated albumin by the nitroblue tetrazolium colorimetric method.

A method has been developed for the measurement of glycated albumin (albumin-fructosamine) by the nitroblue tetrazolium (NBT) colorimetric method. In ...
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