Aug 5, 1985 - Schier, Robert G. Moses,1 and Ignatius E. T. Gan. We presentan improvedfructosamineassay for use withthe. TechniconHA-i 000 analyzer.
I (1985)
CLIN. CHEM. 31/12, 2005-2006
Improved Estimation of Fructosamine, as a Measure of Glycated Serum Protein, with the Technicon RA-1000 Analyzer Fernando
San-GIl,
Gary
M.
Schier,
Robert G. Moses,1 and Ignatius E. T. Gan
We presentan improvedfructosamineassay for use withthe TechniconHA-i 000 analyzer. Featuresof thisassay include: specimen throughput of 100 per hour, within-batchCV of 1.5%, between-batchCV of 2.4%, standardcurve linearupto 24.9 mmoloffructosamineper liter,and goodcorrelation(r = 0.82) with hemoglobinAl. AddItional Keyphrases: diabetes reference inter,al rum-based vs albumin-based standards
__
Measurement of glycated protein is well established as an index of glycemic control. Ordinarily, this has involved the estimation of glycated hemoglobin (1,2), although methods for estimating glycated serum protein are receiving greater attention, e.g., the thiobarbituric acid method (3) and afilnity chromatography (4). These methods, however, are technically demanding, time consuming, and unreliable (3) and offer little advantage over measurement of glycated hemoglobin. A novel approach to measuring glycated serum protein (generically termed fructosamine) that overcomes these problems is the assay developed by Johnson et al. (5). This assay is based on the reducing abffity of ketoamines formed from the Amadori rearrangement of the nonenzymatic condensation products of glucose and protein at alkaline pH. We describe here an improved fructosamine assay adapted for use with the Technicon RA-1000 analyzer.
Materials and Methods Apparatus. Instruments Reagents
We used an RA-1000
analyzer
Corp., North Ryde, NSW 2113). and standards. Nitro blue tetrazolium
(Technicon
Subjects. The diabetic group consisted of 27 Type I and Type U diabetic hospital outpatients. The nondiabetic group consisted of 50 male and 38 female nondiabetic blood donors. Procedures. Serum specimens were collected and stored at -20 #{176}C before analysis. Fructosamine was assayed with the Technicon RA-1000 analyzer by incubating 30 zL of serum for 5 mm with 350 L of the carbonate buffer; 50 pL of the tetrazolium reagent was then added, and the absorbance at 550 nm measured 1 and 2 mm later (Table 1). The absorbance change during this period was used to compare the standard curves prepared with DMF in a serum and albumin matrix. For determination of fructosamine in the subjects the assay was calibrated with a pooled serum solution containing 6.8 mmol of fructosamine per liter (established by assaying standard additions of DM1’). Hemoglobin Al was determined by ion-exchange chromatography (hemoglobin Al test from Bio-Rad, Clinical Division, Epping, NSW 2121).
Results Plotting absorbance change vs concentration and serum-based DMF standards gave two distinct straight lines of best fit: for the latter a slope of 0.0136, y-intercept of 0.0423 A, and r = 0.998;for the former a slope of 0.0203, y-intercept of 0.0317 A, and r = 0.999. Neither line passed through the origin because there was fructosamine in the pooled serum (2.7 mmol/L) or albumin (1.4 mmolfL). Adjusted for these contents, the serum DMF standard curves passed through the origin and were linear to 24.9 mmol/L. We found no difference in results obtained on using, as Linearity.
for the albumin-
(Sigma
Chemical Co., St. Louis, MO 63178), 2.0 mmolfL, was Table 1. RA-1000 Parameters dissolved in carbonate buffer (0.1 mollL, pH 10.35 at 25 #{176}C) Assay and stored in the dark at 4#{176}C. Two sets of standards were prepared by dissolving 0, 1.3, 2.5, 5.0, 10.0, 13.7, and 27.4 mg of 1-deoxy-1-morpholino-nType of analysis % sample volume fructose (DMF, a stable, synthetic Amadori rearrangement Filterposition product), prepared as described by Hedge and Rist (6), and Delay diluting to volume with either pooled human serum or a 40 Incubation g/L solution of human albumin (crystallized, lyophilized First reagentvol (carbonate buffer) human albumin; Sigma) in 140 mmol/L saline, to give final Second reagentvol (tetrazolium) DMF concentrations of 0, 1.0, 2.0, 4.0, 8.0, 11.0, and 22.0 Units Unit factor mmol/L. These were divided and stored at -20 #{176}C.
for Fructosamlne
30
WL550 1 mm 2 mm 70% 5
10% 4 mmol/L 1.000
Decimal point
Department of Biochemistry, The Wollongong Hospital, Crown St., Wollongong,NSW 2500, Australia. ‘Present address: Suite 4, 393 Crown St., Wollongong, NSW 2500, Australia. Received March 19, 1985; accepted August 5, 1985.
Reagentblanklow
1.0
Reagentblank high Slope Intercept Unearity factor 1st limit
2.0 1.0 0.000
1.17 0.0050
CLINICAL CHEMISTRY, Vol. 31, No. 12. 1985
2005
Table 2. Analytical Recovery Data for Serum- and Albumin-Based DMF Standard Curves OMFadded, Sorum-based stds.
mmol/L x
SD
I
SD
1.08 0.10
1.20 0.12
2.08
2.35 4.35
Recovery, 111
0.17 0.30
113 113
8.08 0.52 11.55 0.93 11.83 0.88
105
0.15
3.85 0.31 7.68 0.63
102
Albumin -based
%
x
SD
stds.
Recovery, %
0.81 0.09
75
1.56 0.12 2.91 0.20 5.41 0.37 7.94 0.61
75
76
71 69
starting materials in preparing the matrix, crystallized lyophilized albumin and solutions of human albumin. The latter gave a slope of 0.0205, y-intercept of0.0150A, and r = 0.998. Accuracy and analytical recovery. We added increasing amounts of DMF to 5-mL serum aliquots from four subjects and assayed to determine analytical recovery (Table 2). For the serum-based DMF standards the mean recovery was 109%; for the albumin-based DMF standards it was 73%. Precision. Within-assay and between-assay precision was determined for three concentrations of fructosamine on 14 days (Table 3). Interference. We determined the effect of hemolysis (hemoglobin in concentrations up to 3.0 g/L), bilirubin (up to 166 zmol/L), ascorbic acid (up to 1.2 mmoIJL), and glucose (up to 100 mmol/L) by assaying appropriate dilutions of each in pooled serum. In addition, ascorbic acid interference was determined with and without a 5-mm pre-incubation. Bilirubin and glucose did not interfere at the concentrations tested. Hemoglobin and ascorbic acid interfered significantly by their tetrazolium-reducing activity. Pre-incubation in carbonate buffer for 5-mm decreased this interference greatly. Comparison of fructosamine with hemoglobin Al. Comparison of measured fructosamine concentration (y) with hemoglobin Al concentration (x) in serum from 27 diabetic patients gave a slope of 0.26, y-intercept of 0.88 mmol/L, and r 0.82. Normal reference interval. Values for fructosamine concentration in sera from 50 men and 38 women (blood donors) were normally distributed. The range for the men was 2.0 to 2.8 mmol/L (ii = 2.4, 1.96 SD = 0.43), for the women 2.0 to 2.6 mmolJL (if = 2.3, 1.96 SD = 0.32).
=
Discussion The simplicity and rapidity of the fructosamine assay as compared with other established methods warrants its further development and validation. The assay as performed with the Technicon RA-l000 analyzer has several improvements over previously described methods (5, 7). Its high throughput of 100 specimens per hour greatly exceeds that offered by other methods. Reaction time, including the 5mm pre-incubation to decrease interference by ascorbic acid
Table 3. WIthin- and Between-Assay Precision Fructoumlne, mmoIJL Sample
(5), is only 7 mm. The small sample and reagent requirements make the current cost per assay about 4C (Australian). Finally, the accuracy is increased by using serumbased instead of albumin-based DMF standards, which have different tetrazolium-reducing activities: we obtained significantly different standard curves with the two, and when we used albumin-based DMF standards, the analytical recovery of DMF added to sera was only about 73%, as compared with 109% for serum-based DMF standards. Johnson et al. (5) also demonstrated the different nitro blue tetrazoliumreducing activities of albumin and serum, but authors of later reports (7, 9-12) continue to use albumin. Differences in reported reference intervals are attributable to the use of serum-based or albumin-based DMF standards. In a recently released commercial assay for fructosamine (Roche Diagnostica) the method described by Johnson et al. (5) is used, but the reference range listed (2.0 to 2.8 mmoIJL) is higher than those reported previously (7, 9-12) by this method. There was no interference by biirubin and glucose in respective concentrations up to 166 /LmoIJL and 100 mmol/L. Hemolysis and ascorbic acid interference was significantly diminished by pre-incubating the serum specimens in carbonate buffer before adding the tetrazolium reagent; this may also decrease the reducing activity of other interfering agents such as glutathione. The development of this rapid, low-cost, and reproducible method, the results of which correlate well (r = 0.82) with values for hemoglobin Al, may lead to its increased use in hospital and medical practice.
Funding was provided by the illawarra Diabetic Research Fund. Grants from Lilly Industries Pty. Ltd. and the Wollongong Diabetic Association were made to the fliawarra Diabetic Research Fund. We also gratefully acknowledge the technical assistance of Anthony Elderfield, Robert Glen, and Ian Whitley.
References 1. Moses R, Bowen G, Edwards R. Fastmoving hemoglobin:A new
index of diabetic control. Aust J Med Technol 10, 153-156 (1979). 2. Miedema K, Casparie T. Glycosylated hemoglobins: Biochemical evaluation and clinical utility. Ann Clin Biochem 21, 2-15 (1984). 3. Dolhofer R, Wieland OH. Improvement of thiobarbituric acid assay for serum glycosylprotein determination. Clin Chim Acta 112,
197-204 (1981). 4. Schleicher ED, Gerbitz KD, Doihofer R, et al. Clinical utility of nonenzymatically glycosylated blood proteins as an index of glucose control. Diabetes Care 7,548-556(1984). 5. Johnson RN, Metcalf PA, Baker JR. Fructosamine: A new approach to the estimation of serum glycosylprotein. An index of diabetic control. Clin Chim Acta 127, 87-95 (1982). 6. Hedge JE, Rist CE. The Amadori re-arrangement under new conditions. JAm
Chem Soc 75, 316-322
(1953).
7. Lloyd D, Marples J. Simple colorimetry of glycated serum protein in a centrifugal analyzer. Clin Chem 30, 1686-1688(1984). 8. Dothofer R, Wieland OH. Glycosylation of serum albumin: Elevated glycosyl-albumin in diabetic patients. FEBS Lett 103, 282-286 (1979). 9. Baker JR, O’Connor JP, Metcalf PA, et al. Clinical usefulness of estimation of serum fructosamine concentration as a screening test for diabetes mellitus. Br Med J 287, 863-867 (1983). 10. Roberts AB, Baker JR. Court DJ, et al. Fructosamine in diabetic pregnancy. Lancet ii, 998-999 (1983). 11. Baker JR, Johnson RN, Scott DJ. Serum fructosamine concentrations in patients with type II (non-insulin-dependent) diabetes mellitus during changes in management. Br Med J 288, 1484-1486 (1984).
Moan
SD
Low Medium
2.3 3.4
0.05 0.05
High Between-assay
8.6
0.05
2.2 1.5 0.6
Low
2.2
0.05
23
12. Baker JR, Metcalf PA, Holdaway IM, Johnson RN. Serum
Medium High
3.3 8.6
0.09 0.21
27
fructosamine concentration as a measure of blood glucose control in type I (insulin dependent) diabetes mellitus. Br Med J 290,352-355
Within-assay
2006 CLINICALCHEMISTRY, Vol. 31, No. 12, 1985
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