Elimination of superoxide dismutase interference in fructosamine assay

Clinical Biochemistry, Vol. 32, No. 3, 185–188, 1999 Copyright © 1999 The Canadian Society of Clinical Chemists Printed in the USA. All rights reserved 0009-9120/99/$–see front matter

PII S0009-9120(99)00016-8

Elimination of Superoxide Dismutase Interference in Fructosamine Assay BABU L. SOMANI,1 VIVEK AMBADE, PANDURANG M. BULAKH,2 and YELESWARAPU V. SHARMA1 1

Department of Biochemistry, Armed Forces Medical College and 2B. J. Medical College, Pune 411 040, India

Objective: Superoxide dismutase (SOD) (EC 1.15.1.1) is reported to decrease the reduction of nitroblue tetrazolium (NBT) in the fructosamine assay. The study was undertaken to find a method to eliminate this interference. Design and Methods: We studied the NBT reduction in the presence and absence of added bovine erythrocyte SOD during fructosamine assay. Formation of reduced NBT decreased with the increasing concentration of SOD. Various inhibitors of SOD were experimented with for effectively eliminating this interference. Results: Cyanide eliminates the interference due to SOD, but is unsuitable because in it’s presence glucose becomes reducing under the conditions of fructosamine assay. SOD inhibitors such as EDTA and Azide did not eliminate the effect of SOD. Guanidine. HCl gives opalescence in the reaction mixture. Addition of 2M HCl to the serum and incubation at 37 °C for 10 min eliminated the effect of added SOD (70 kU/L).The correlation between dA10 –20 min of serum treated with HCl in presence and absence of added SOD is y 5 0.9011x 1 0.01055 with r 5 0.9789 and S.E. (y) 0.007686. Conclusion: SOD does not interfere in globin bound fructosamine assay as acid-acetone treatment in preparation of heme free globin inhibits SOD. Pretreatment of serum with HCl can satisfactorily eliminate the interference due to SOD in fructosamine assay. The acid treatment could be used to inhibit SOD in various other reactions that are followed with NBT reduction. Copyright © 1999 The Canadian Society of Clinical Chemists

KEY WORDS: fructosamine; glycated hemoglobin; superoxide dismutase (SOD); SOD inhibitor.

Introduction erum fructosamine assay is a simple colorimetric test that measures glycated serum protein concentration (1,2). We additionally modified the reagent by including p-chloro mercuribenzoic acid (pCMB) in the reaction mixture to bind free-SH groups so that the same reagent could be used to quantify the serum glycated proteins as well as glycated hemoglobin (3). Different mechanisms have been proposed to explain the interference due to the presence of SOD in estimation of serum fructosamine. Jones et al. (4) speculated that superoxide

S

Correspondence: B.L. Somani, Scientist-F, Department of Biochemistry, Armed Forces Medical College, Pune 411 040, India. E-mail: [email protected] Received October 7, 1998; revised February 9, 1999; accepted February 12, 1999. CLINICAL BIOCHEMISTRY, VOLUME 32, APRIL 1999

radical produced as an intermediate in the oxidation of glycated albumin mediates the NBT reduction, while Baker et al. (5) proposed that superoxide is not an intermediate and the inhibitory effect of SOD and catalase is probably the result of cyclic oxygen generation in the assay mixture reducing the formation of reduced NBT. The increased and variable contents of SOD have been reported in the serum from certain cases of diabetes mellitus (6). Kruse-Jarres et al. (7) suggested incorporation of non-ionic surfactant and uricase. The surfactant mediated effect on inhibition of SOD was due to peroxides present in detergent that inhibit SOD and eliminate the interference (5), but suffer from the drawback of variable concentration and nonspecific effect on NBT reduction. Inclusion of SOD inhibitors such as cyanide and azide was suggested to eliminate the interference due to SOD (5). Therefore, we investigated the effect of various SOD inhibitors on fructosamine assay at SOD activity of 30 –70 kU/L, which is in the upper normal to high range (normal 20 –57 kU/L) (8). Materials and methods Nitro blue tetrazolium (NBT), p-CMB, and SOD (Bovine erythrocytes) were from Sigma Chemical Co. (St. Louis, MO, USA). Other reagents and chemicals used were of analytical grade from the local sources. The blood samples were obtained from diabetic (n 5 8) and non-diabetic (n 5 10) patients reporting to out-patient department (OPD). Specimens were allowed to clot and serum was separated and either used fresh or stored at 4 °C for 1–2 days. The fructosamine assay was carried out with NBT reagent containing p-CMB (3). Serum (0.1 mL) was treated with equal volume of inhibitor followed by bicarbonate buffer to make the volume equal to 700 mL and NBT reagent (700 mL). Reaction mixture was incubated at 37 °C and the absorbance was measured at 530 nm at 10 and 20 min in Shimadzu spectrophotometer CL-750. The dA10 –20 min (dA) was calculated. 185

SOMANI

ET AL.

TABLE 1 Effect of Various Inhibitors on Interference of SOD in Fructosamine Assay SOD Inhibitor (Concentration)a None (serum) HCl (0.014 M) EDTA (1 mM) Azide (5.5 mM) Cyanide (15 mM)b Heat (65 °C, 30 min, saline) Heat (65 °C, 30 min, pH 10.35) Guanidine.HClc (0.44 M) Globin (sample)

DA Sample

DA Sample and SOD (30 kU/L)

% Change in DA

0.123 0.121 0.104 0.105 0.414 0.125

0.074 0.120 0.063 0.065 0.414 0.072

240.0 21.0 239.5 238.1 0.0 242.4

0.176

0.176

0.0

ppt 0.186

ppt 0.187

— 0.0

a

Final concentration in the reaction mixture. The inhibitor is added in equal volume to the serum. b Serum glucose concentration was 2.22 mmol/L. c Guanidine.HCl precipitates on addition of reagent.

STUDY

Results

OF

SOD

INTERFERENCE ON GLOBIN BOUND

FRUCTOSAMINE ESTIMATION

EFFECT

OF CYANIDE AND OTHER

SOD

INHIBITORS

We studied the effect of cyanide in the reaction mixture. It was observed that inclusion of CN2 in the assay mixture at final concentration of 15 mM increased the dA (Table 1). Cyanide causes the inhibition of SOD as the dA in presence and absence of SOD is same. We, subsequently, observed that glucose even at 0.55 mmol/L was reactive and caused the reduction of NBT in the presence of 15 mM CN2, whereas it was not reactive in the absence of CN2. Therefore, the increase in dA in the presence of CN2 and absence of SOD added could be due to the reduction of NBT by glucose. The aldoses such as glucose and ribose were reported not to be reactive in the presence of CN2 toward NBT (9) at pH 8.3; however, in fructosamine reagent glucose reduces NBT in presence of CN2. This may be due to the reagent pH being 10.35 instead of 8.3. The rate of reduction even in case of glyceraldehyde has been reported to increase greatly with increase in pH (9). We studied the effect of inhibitors of SOD such as azide and EDTA. The dA in presence of these inhibitors decreases on addition of SOD (Table 1) and, therefore, these are not effective. This could be due to the fact that NBT reduction is studied in bicarbonate buffer of pH 10.35, and these inhibitors may not be active in this condition. Guanidine.HCl got precipitated on the addition of NBT reagent therefore, its effect on inhibition of SOD could not be evaluated. Heating at 65 °C for 30 min in the presence of bicarbonate buffer (pH 10.35) inhibited SOD but glucose also reduces NBT under these conditions and the dA becomes artificially high. Heating at 65 °C in saline for 30 min did not inhibit SOD (Table 1). 186

We also investigated whether the estimation of glycated hemoglobin as globin bound fructosamine using NBT reagent is affected by SOD present in RBC or not. We added SOD to the globin solution and observed that the rate of NBT reduction is same for globin solution with and without added SOD (Table 1), therefore, indicating that SOD does not interfere. Residual heme or acetone were contemplated to be the cause for this inhibition of SOD. However, when acetone supernatant containing heme was added to the serum sample containing SOD, the effect of SOD was not eliminated. The pH of the globin solution was found to be less than 2.0, probably because globin precipitate binds to the HCl present in acid-acetone mixture and gives an acidic solution when dissolved. Considering this low pH as cause of denaturation and inactivation of added SOD, we thought it worthwhile to experiment with HCl in serum fructosamine assay. EFFECT

OF

HCl

ON

SOD

INTERFERENCE IN SERUM

FRUCTOSAMINE ASSAY

Serum sample supplemented with SOD was treated with HCl, the acid was subsequently neutralized with equimolar NaOH. The change in dA was found to be same for serum with and without added SOD (Table 1), therefore, eliminating the effect of added SOD on inhibition of NBT reduction. The conditions were standardized to eliminate SOD interference. The test sample containing 100 mL of serum/serum containing SOD was added with 100 mL of 2N HCl. Although HCl is found to inhibit SOD within 1 min, we incubated this for 10 min at 37 °C. This was followed by addition of 300 mL of carbonate buffer (pH 10.35) and 1M NaOH (200 mL) to neutralize the added HCl. The HCl and NaOH CLINICAL BIOCHEMISTRY, VOLUME 32, APRIL 1999

SUPEROXIDE DISMUTASE INTERFERENCE

Figure 1 — The effect on fructosamine assay of different concentration of SOD added and its inhibition by HCl.

were first titrated and adjusted to neutralize each other. To this reaction mixture equal volume of NBT reagent (700 mL) containing p-CMB as described by Somani et al. (3) was added. The dA10 –20 min (dA) was observed at 530 nm. The control samples were similarly processed but the order of addition of reagents was serum followed by buffer, HCl, and NaOH without any incubation step in between. The addition of buffer before addition of HCl prevents lowering of pH and, therefore, inhibition of SOD. The precision of assay is not significantly affected as mean dA of replicate analysis (n 5 10) is 0.125 and 0.119 for HCl treated and control sample with CV of 2.27 and 2.07%, respectively. The effect of added SOD and its inhibition by HCl on fructosamine assay as measured by reduction of NBT is given in Figure 1. The addition of SOD causes reduction in dA. The percentage of NBT reduction decreases with increasing concentration of SOD, addition of HCl eliminates this decrease in dA. Figure 2 shows the correlation between values of dA of various serum samples against serum samples

supplemented with SOD (70 kU/L) in presence and absence of HCl treatment to inhibit SOD. The correlation coefficient (r 5 0.9789) and y 5 0.9011x 1 0.01055 indicates that the effect of added SOD (70 kU/L) is completely eliminated by addition of HCl. Moreover, no significant effect on intercept is noticed when dA for serum samples with SOD, are plotted against dA for serum samples without SOD in presence of HCl (Figure 2). The dA values show good correlation even in the absence of HCl as all the samples were supplemented with same amount of SOD. However, it can be observed that dA is reduced in comparison to the serum samples that have been treated with HCl, because the SOD remains active and interferes in fructosamine assay (Figure 2). Discussion Although the cyanide inhibits SOD under the conditions of fructosamine assay it cannot be used to eliminate the interference as glucose becomes reactive and causes reduction of NBT, this is similar to the reported effect of cyanide on the reduction of NBT by a-hydroxyaldehydes (9).The azide, EDTA, and guanidine.HCl are not effective as inhibitors of SOD under the conditions of fructosamine assay. The HCl is not only able to effectively inhibit the added SOD but also does not appear to cause any nonspecific increase in the dA as observed with detergents where the intercept in the correlation curve is reported to be increased (5), which indicates significant nonspecific effect of detergent on fructosamine assay. The use of HCl as an inhibitor of SOD will also not have the problem of variability in the concentration of inhibitor, as found in the case of peroxides present in detergents. Therefore, modification of fructosamine assay with use of HCl to inhibit SOD could make the fructosamine assay more specific. References

Figure 2 — Effect of SOD (70 kU/L) added to patient samples in fructosamine assay. Correlation between dA of sera with and without added SOD before and after treatment with HCl. The sera were obtained from euglycemic (n 5 10) and hyperglycemic (n 5 8) individuals. The untreated sera were processed similarly, changing the order of buffer and HCl. CLINICAL BIOCHEMISTRY, VOLUME 32, APRIL 1999

1. Johnson RN, Metcalf PA, Baker JR. Fructosamine: A new approach to the estimation of serum glycosyl protein. An index of diabetic control. Clin Chim Acta 1982; 127: 87–95. 2. Baker JR, Metcalf PA, Johnson RN, Newman D, Rietz P. Use of protein based standards in automated colorimetric determinations of fructosamine in the serum. Clin Chem 1985; 31: 1550 – 4. 3. Somani BL, Sinha R, Gupta MM. Fructosamine assay modified for the estimation of glycated hemoglobin. Clin Chem 1989; 35: 497. 4. Jones AF, Winkles JW, Thornalley PJ, Lunec J, Jennings PE. Inhibitory effect of superoxide dismutase on fructosamine assay. Clin Chem 1987; 33: 147–9. 5. Baker JR, Zyzak DV, Thorpe SR, Baynes JW. Mechanism of fructosamine assay: Evidence against role of superoxide as intermediate in nitroblue tetrazolium reduction. Clin Chem 1994; 40: 1950 –5. 6. Kawagachi T, Suzuki K, Matsuda Y, et al. Serum manganese-superoxide dismutase: Normal values and increased levels in patients with acute myocardial infarction and several malignant diseases determined 187

SOMANI

by an enzyme-linked immunosorbent assay using a monoclonal antibody. J Immunol Methods 1990; 127: 249 –54. 7. Kruse-Jarres JD, Jarausch J, Lehmann P, Vogt BW, Rietz P. A new colorimetric method for the determination of fructosamine. Lab Med 1989; 13: 245–53. 8. Guemouri L, Artur Y, Herbeth B, Jeandel C, Cuny G, Siest G. Biological variability of superoxide dis-

188

ET AL.

mutase, glutathione peroxidase, and catalase. Clin Chem 1991; 37: 1932–7. 9. Robertson P, Fridovich S, Mishra H, Fridovich I. Cyanide catalyzes the oxidation of a-hydroxyaldehyde and related compounds: Monitored as the reduction of dioxygen, cytochrome-C and nitroblue tetrazolium. Arch Biochem Biophys 1981; 207: 282–9.

CLINICAL BIOCHEMISTRY, VOLUME 32, APRIL 1999