and glycat- ed protein. The reagent for the fructosamine assay was sodium carbonate buffer. (100 mmolIL,. pH. 10.35) containing. 0.25 mmol of NBT per liter.
LIN.
CHEM. 33/1,
Inhibitory Ian
147-149
(1987)
Effect of Superoxide
F. Jones,’
John W. Winkles,1
Dismutase
Paul J. Thornalley,2
The fructosamine assay measures the degree matic protein glycation by virtue of the reducing
analytical
changes
significance
in serum
of nonenzyproperties of
ddItIonal
pathological
significance
marked
on the glycated in vitro and of intermediacy in the I rucintermediates may be of
anti-oxidant activity
be of pathological and protein
when
Lunec,’
Joseph
such proteins in alkaline conditions. We report the inhibitory effect of superoxide dismutase (EC 1.15.1.1)
reducing activity both of protein diabetic sera, indicating superoxide tosamine reaction. The free-radical
on Fructosamine
and
physiological
occur. They may also
in diabetic
microangiopathy
browning.
Keyphrases:
free radical
diabetes
mel/it us
glycated
proteins
intermediates
Nonenzymatic protein glycation is clinically important in the assessment of glycemic control in diabetes mellitus (1) and may be implicated in the pathogenesis of diabetic rnicroangiopathy (for a review see ref.2). Several methods are available for measuring the degree of glycation of hemoglobin (3) and of serum and tissue proteins (4). Ketoantines, also known generically as fructosamines, are Lormed by an Amadori rearrangement of the initial Schiff base adduct of glucose with protein amino groups (5). Fructosamines are reductants under alkaline conditions (6), property used in the colorimetric assay of serum protein lycation, in which such proteins reduce the dye nitroblue etrazolium (7). Concentrations of fructosamines so meaured correlate well with other indices of glycemia (8). The may is precise, cheap, and readily automated. Although the reducing activity of glycated protein deends on the presence of ketoamines, the exact nature of the eductant involved is unclear. Here we present evidence iat the fructosamine assay depends on the production of uperoxide radical by glycated protein.
Assay
Paul E. Jennings,3
and Anthony
Incubations. Bovine lin, dissolved at a phosphate-buffered glucose (50 mmolJL) h, then dialyzed for to remove
free
H. Bamett3
albumin and human gamma-globuconcentration of 40 g/L in isotonic saline, pH 7.4, were incubated with under sterile conditions at 37 #{176}C for 48 24 h against phosphate-buffered saline
glucose.
Measurement of the reducing activity of serum and glycated protein. The reagent for the fructosamine assay was sodium carbonate buffer (100 mmolIL, pH 10.35) containing 0.25 mmol of NBT per liter. The assay was performed at 37#{176}C, in a centrifugal analyzer (“Encore”; Baker Instruments Ltd., Egham, Surrey, U.K.). We automatically dispensed 250 pL of reagent, 20 L of sample, and 60 jL of water as diluent, using a “Pipettor 1000” (Baker Instruments Ltd.). The rate of reduction of NBT was measured kinetically by monitoring the absorbance change at 520 nm, after a 6-mm preincubation. In the inhibition experiments, superoxide dismutase and ceruloplasmin were dispensed manually as 20-pL aliquots into the reagent compartment of the analyzer disc. Standards for the reaction were DMF dissolved in human albumin solution. The CV for the assay was 1.5% within batch and 3% between batch. Results We found that the reduction of NBT by both albumin and gamma-globulin that had been glycated in vitro, and by sera from diabetics, was markedly inhibited by added superoxide dismutase (Figure 1). We assayed the diabetic serum pool both before and after dialysis against phosphate-buffered saline to eliminate low-molecular-mass reductants, notably vitamin C (7). The reducing activity of the undialyzed diabetic serum pool was less susceptible to inhibition by superoxide dismutase (Figure 1) than the activity of either 25
5
r
laterlals
and Methods
Superoxide dismutase (bovine, EC 1.15.1.1), aruloplasmin (human, type X), bovine serum albumin, uman gamma-globulin (Cohn Fraction II), 1-deoxy-1-morholinofructose (DMF), and nitroblue tetrazolium (NBT) ‘ere all purchased from Sigma (London) Chemical Co.,
4
Materials.
ingston,
Surrey,
U.K.
Human
albumin
solution
(45
g/L)
‘as obtained from the National Blood Transfusion Service, lood Products Laboratory, Elstree, Herts., U.K. All other hemicals were of “ANA1.a” grade, from BDH Ltd., Poole, lorset, U.K. Blood samples. Blood was sampled from 29 diabetic
20 C
r 0
3
15
2
10
Ui C 0
0
0
C
0
ubjects
attending
the outpatient oth separately and as a pool.
clinic.
Sera
were
1 Department of Biochemistry, Selly Oak Hospital, oad, Selly Oak, Birmingham B29 6JD, U.K. 2
Department
of Pharmaceutical
Sciences,
University
assayed
Raddlebarn
Birmingham B4 7ET, U.K. of Medicine, University of Birmingham and East irmingham Hospital, Bordesley Green, Birmingham B9 5ST, U.K. Received August 18, 1986; accepted October 10, 1986.
ston Triangle, Department
0.05
0.1
LOGJ
of Aston,
0.5
1
SUPEROXIDE
10
DISMUTASE
100
MG/L
Fig. 1. Effect of superoxide dismutase on the reducing activity diabetic sera and glycated albumin towards nitroblue tetrazolium -U, pool;
of
undialyzed pooled serum from diabetics; LJ-L], dialyzed diabetic serum glycated albumin, 40 g/L. Results are expressed as mean ± 1 SD
A-A,
CLINICAL CHEMISTRY,
Vol. 33, No. 1, 1987
147
the dialyzed serum pool or glycated albumin, whose residual reducing activities at maximal inhibition by superoxide dismutase were similar (22% and 15%, respectively). Nevertheless, most of the reducing activity of individual undialyzed diabetic sera was inhibited by superoxide dismutase (100 mgtL) to a mean residual activity of 47% of the original (n = 29) (Figure 2). Ceruloplasmin, which has a minor superoxide-radical scavenging activity (9), also inhibits the reducing activity of glycated albumin (Figure 3), though the effect
is less pronounced
than
that
of superoxide
dismutase. +
DIscussion radical, O2, produced by a single-electron of molecular oxygen, has both reducing and oxidizing properties (10), although at the pH of the fructosamine assay the radical is predominantly a reductant. The radical has a short half-life but can be assayed by its ability to reduce suitable dyes, such as NBT, in the presence and absence of superoxide dismutase. Superoxide dismutase catalyzes the dismutation of superoxide to oxygen and hydrogen peroxide, and has maximal enzymatic activity at alkaline pH (11). Reducing activity that can be inhibited by superoxide dismutase is attributed to the superoxide radical, and this principle is used in a standard assay for superoxide radical (12). The
superoxide
reduction
6
5 -J -J
0
4
CO
I-
600 CAERULOPLASMIM
000 tIG/L
1000
Fig. 3. Effect of ceruloplasmin on the reducing actMty albumin towards nitroblue tetazolium Results are expressed as mean ± 1 SD
1200
1400
of glycatec
n=29
sufficiently high to inhibit the fructosamine assay som.. what. Similarly, ceruloplasmin concentrations in serum ar markedly elevated in pregnancy, with estrogen therap: and in association with inflammatory conditions (15). Thu pathological and physiological changes in diabetic sera tha cause an increased superoxide-radical scavenging activit may interfere analytically with the fructosamine assa: This could mask poor diabetic glycemic control if it wez assessed by the fructosamine method alone. The free-radical basis of the reducing activity of nonenz
r=O.94
matically
.
C.,
3 U-J
II-
2
1
glycated
protein
may
be
of direct
pathologic
significance in the pathogenesis of the chronic complicatior of diabetes mellitus. Nonenzymatically glycated protein undergo browning reactions in which they develop cros links and fluorescence, and such changes have been corn
1 SOD-RESISTANT Fig. 2. Relation
CLINICAL
between
2 FRUCTOSAMINE
3 MMOL/L
of fructosamine and superoxide dismutase-resistant fructosamine in serum from diabetics Fructosamine concentrations were measured in 29 undialyzed diabetic sera, with and without added superoxide dismutase (100 mg/L). y = 1 .85x + 1.45 mmol/L 148
400
The effect of superoxide dismutase on the reducing activi ty of glycated protein and diabetic sera towards NB’I indicates superoxide intermediacy in this reaction. W propose that the ketoamine, or its eneaminol tautome: reduces molecular oxygen in a one-electron transfer t generate superoxide radical, which in turn reduces NBT. single electron autoxidation of the ketoamine would neces sarily generate a free-radical intermediate of the glycate protein. Although the nature of this latter radical is un known, by comparison with the autoxidation of monosaccha rides (13) it may be an eneaminoxy radical of the glycate protein (Figure 4). This mechanism may be of analytical significance shoul the antioxidant activity of serum be altered. Although th normal concentration of superoxide dismutase in serum i too low (14) to interfere with the assay, the enzyme accumi lates in renal impairment, and its activity could then 1
7
w
200
concentrations
CHEMISTRY,
Vol. 33, No. 1, 1987
lated with the presence of microangiopathy (for a review se. ref. 16). Browning depends on the further reaction of th ketoamine adduct, probably the oxidation of the eneanlinl reductones, and occurs more rapidly under alkaline cond tions (17, 18). We have shown that this oxidation of th eneaminol involves superoxide radical production and likely to involve free-radical intermediates of the glycate protein. The properties of fluorescence (19) and aggregatio
1PTElNf-
3. Miedema
JPROTE1NI-t
IPROTEINI-N H-0
cal evaluation
H
-
-
HCW
(O4)3
(JH)3
O0H
O0H
G0H
fiLDIMINE
KZItWIINE
It’ lPR0TElNl-Il
1PRcrE*i1
H
K II
C-c
C-OH
()3 OOH
O2OH
RADICAL
E?#{128}NIIMX.
T. Glycosylated haemoglobins: biochemiutility. Ann Clin Biochem 1984;21:2-15.
4. Kennedy
L, Mehl TD, Riley WJ, Merimee TJ. Non-enzymatically glycosylated serum protein in diabetes mellitus: an index of short term glycaemia. Diabetologia 1981;21:94-8.
5. Buns HF, Haney DV, Gabbay KH, et al. Further identification of the nature and linkage of the carbohydrate in haemoglobin A1. Biochem Biophys Res Commun 1975;67:103-9. 6. Hodge JE. The Amadori rearrangement. Adv Carbohydr Chem 1955;10:169-205. 7. 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. 8. 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 1983;287:863-7. 9. Goldstein IM, Kaplan HB, Edelson HS, Weissman G. Caeruloplasmin, a scavenger of superoxide anion radical. J Biol Chem 1979;254:4040-5.
10. Halliwell EWNIIWL
K, Casparie and clinical
medicine.
B, Gutteridge JMC. Free radicals in biology Oxford, U.K.: Oxford University Press, 1985:57-63.
and
11. McCord JM, Fridovich I. Superoxide dismutase, an enrymic function for erythrocuprein. J Biol Chem 1969;244:6049-55. 12. Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem SUFIDE
RADICAL
2
> NBT
Fig. 4. Possible mechanism the fructosamine reaction
for the involvement
of superoxide
radical in
which are characteristically teins, are also typical of proteins
observed in browned prothat have been subject to free-radical-mediated oxidative damage (21). We suggest that protein ketoamines may, by a process of slow autoxidation under physiological conditions, generate free-radical intermediates and thereby contribute to this browning reaction. (20),
References 1. Dunn PJ, Cole TA, Soeldner JS, et al. Temporal relationship of glycosylated haemoglobin concentrations in glucose control in diabetes. Diabetologia
1979;17:213-20.
2. Kennedy L, Baynes chronic complications 1984;26:93-8.
JW. Non-enzymatic of diabetes: an
glycosylation and the overview. Diabetologia
1971;44:276-87. 13. Thornalley PJ. Monosaccharide autoxidation in health and disease. Environ Health Persp 1985;64:297-307. 14. Marklund SL, Holme E, Heilner L. Superoxide dismutase in extracellular fluids. Clin Chim Acta 1982;126:41-51. 15. Cox DW. Factors influencing serum caeruloplasmin levels in normal individuals. J Lab Olin Med 1966;68:893-904.
16. Anon. Browning and diabetic complications. [Editorial]. Lancet 1986;i:1192-3. 17. Hedge JE, Rist CE. The Amadori rearrangement under new conditions and its significance for non-enzymatic browning reactions. J Am Chem Soc 1952;75:316-22. 18. Eichner K. Antioxidative effect of Maillard reaction intermediates. In: Simic MG, Karel M, eds. Autoxidation in food and biological systems. New York: Plenum Press, 1980:367-85. 19. Monnier VM, Vishwanath V, Frank KE, Elmets KE, Daughot P, Kohn ER. Relation between complications of type 1 diabetes mellitus and collagen linked fluorescence. N Engl J Med 1986;314:403-8. 20. Schnider S, Koim RR. Effects of age and diabetes mellitus on the solubility and non-enzymatic glucosylation of human skin collagen. J Olin Invest 1981;66:1630-5. 21. Lunec J, Blake DR. McCleary SJ, Brailsford S, Bacon P. Self. perpetuating mechanisms of immunoglobulin G aggregation in rheumatoid inflammation. J Clin Invest 1985;76:2084-90.
CLINICAL
CHEMISTRY,
Vol. 33, No. 1, 1987
149
