ISSN 0306-3623/98 $19.00 1 .00
PII S0306-3623(98)00016-0
All rights reserved
Gen. Pharmac. Vol. 31, No. 3, pp. 441–445, 1998
Copyright  1998 Elsevier Science Inc.
Printed in the USA.
Reducing Sugars
Trigger d-Aminolevulinic Dehydratase
Inactivation: Evidence of In Vitro Aspirin Prevention*
Fabiana A. Caballero, Esther N. Gerez,
César F. Polo, Elba S. Vazquez and Alcira M. del C. Batlle†
Centro de Investigaciones sobre
Porfirinas y Porfirias (CIPYP), (CONICET—FCEN, UBA),
Ciudad Universitaria, Pabellón II, 2do Piso, 1428 Buenos Aires, Argentina
ABSTRACT. 1. The effect of in vitro glycation on d-aminolevulinic dehydratase (ALA-D) under
several experimental conditions was investigated. When preincubated with 500 mM glucose at 378C for
20 hr, ALA-D was 80% inactivated and glycated hemoglobin levels were increased more than fourfold.
2. Thiobarbituric acid species were not modified during glycation; therefore ALA-D inactivation
cannot be attributed to glucose autoxidation.
3. Acetyl salicylic acid was effective in preventing both hemoglobin glycation and ALA-D inactivation by glucose.
4. A method has been developed for measuring protein glycation in vitro, in a crude preparation of
red blood cells, which can also be applied to sugars other than glucose. gen pharmac 31;3:441–445,
1998.  1998 Elsevier Science Inc.
KEY WORDS. Glycation, carbohydrates, acetyl salicylic acid, lipid peroxidation, d-aminolevulinic
dehydratase, enzyme inactivation, glycated hemoglobin
INTRODUCTION
Nonenzymatic glycation of proteins, mainly hemoglobin, and the
relation between this process and diabetes mellitus has been extensively reviewed (Strowig and Raskin, 1995). The aldehyde group of
glucose is known to react with amino groups of proteins, resulting
in a Schiff-base conjugate formation, which subsequently undergoes
an Amadori rearrangement leading to a stable ketoamine adduct
(Nagaraj et al., 1996).
Glycation is the first of a series of reactions known as “Maillard
browning” (Nagaraj et al., 1996). This process can promote in vivo
and in vitro cross-linking of proteins through e-NH2 groups of lysine
or hydroxylysine. Maillard browning is enhanced in vivo in the diabetic condition, especially in tissues in which protein turnover is
slow and physiological effects are stronger than in blood, such as in
lens crystallin (Thorpe and Baynes, 1996) and tendons (Kochakian
et al., 1996). There is also some evidence suggesting that protein
glycation may be implicated in the long-term complications of diabetes (Brownlee, 1992; Cerami et al., 1991), and in aging diseases
(Thorpe and Baynes, 1996), and it is the primary causal factor for
the development of diabetic microvascular complications (Brownlee,
1995). It is known that protein cross-linking may either alter protein functions or change their susceptibility to enzymatic degradation (Fu et al., 1994); moreover, in vivo and in vitro modifications in
enzymatic activities have been well documented (Brownlee, 1994).
On the other hand, it has been demonstrated that aspirin can prevent in vitro and in vivo glycation of albumin and hemoglobin by
acetylation of the potential glycation sites (Rendell et al., 1986).
*To the memory of our beloved César.
† To whom correspondence should be addressed, at Viamonte 1881 108
“A,” 1056 Buenos Aires, Argentina [Fax: (541)-811-7447; E-mail: cipyp@
alad.fcen.uba.ar].
Received 5 September 1997; accepted 15 December 1997.
Studies carried out in a diabetic population showed that the activities of several heme enzymes such as d-aminolevulinic dehydratase
(ALA-D), porphobilinogen deaminase and uroporphyrinogen decarboxylase are diminished in blood (Caballero et al., 1995). Alterations in the heme pathway were also observed in streptozotocininduced diabetic mice (Polo et al., 1995).
The aim of this study was to investigate the effect of in vitro glycation on ALA-D under different experimental conditions; the evolution of this process was correlated with the amount of glycated hemoglobin produced. To determine if lipid peroxidation products can
promote protein glycation, the peroxidation index was estimated.
Furthermore, the potential role of acetyl salicylic acid (ASA) (aspirin) in preventing the nonenzymatic glycation was examined.
MATERIALS AND METHODS
Materials
All chemicals used were reagent degree and obtained from Sigma
(St. Louis, MO, USA).
Enzymatic source
Blood from healthy volunteers was obtained by venipuncture, withdrawn in a heparin- or EDTA-containing flask and kept at 48C.
Glycation procedure
Red blood cells (RBCs) were separated, washed with saline and preincubated at 378C in the dark and aerobiosis, for as long as 20 hr
in humid atmosphere, with shaking, after adding the sugar at a final
concentration of 500 mM in a balanced salt solution (glucose free),
unless otherwise specified (RBC:balanced salt solution, 1:10 v/v).
442
F. A. Caballero et al.
Controls, without sugar but preincubated in the same conditions,
were included in all experiments. The addition of any carbohydrate
to the enzyme assay mixture during incubation, at a final concentration of 500 mM, had no effect on enzyme activity. Samples of RBCs
were preincubated with or without glucose in the presence of ASA
(10 mM) for as long as 20 hr.
Enzyme assays
ALA-D activity was determined by the method of Batlle et al. (1967).
Because preincubation periods could be as long as 20 hr, zinc acetate
was added to the medium at a final concentration of 1 mM to prevent oxidation of thiol groups. Activity was expressed as units per
milligram of hemoglobin (Hb). An enzyme unit is defined as the
amount of enzyme that catalyzes the formation of 1 nmol of product
under the standard incubation conditions.
Glycosylated hemoglobin (GHb) was measured by using the method
described by Parker et al. (1981) and was expressed as nanomoles of
hydroxymethylfurfural (HMF) per milligram of Hb.
The peroxidation index was estimated by the formation of malondialdehyde (MDA) and determined as thiobarbituric acid reactive
species (TBARS) by the method of Niehaus and Samuelson (1968).
Statistics
Data represent mean values6SD of at least four separate experiments conducted in triplicate and were analyzed statistically by using nonpaired Student’s t-test with the Sigma Plot 2.0 statistical
software for IBM.
RESULTS
Effect of glucose concentration
FIGURE 1. Effect of preincubation in the presence of different
glucose concentrations on ALA-D activity (d) and GHb formation (j). Red blood cells were preincubated for 20 hr at 378C
with glucose (50–500 mM final concentration) or without the
sugar. Other experimental details were as indicated in the text.
*P,0.05. HMF5hydroxymethylfurfural.
the glycation process. GHb levels augmented along the period of
assay; a correlation between ALA-D inactivation and GHb formation was found (Fig. 3B).
The peroxidation index was determined to evaluate if the enzyme
inactivation was produced as a consequence of glucose oxidation. No
ALA-D activity was determined after preincubating RBCs with different glucose concentrations (range, 50–500 mM). Enzyme activity
was diminished by about 10% at the lowest concentration of sugar
assayed; the inhibition profile was sharper for higher glucose concentrations and reached an inhibition of 80% at 500 mM of glucose,
with respect to controls under the same conditions but in the absence of glucose (Fig. 1). The progression of glycation was followed
by the detection of GHb, which augmented more than fourfold at
500 mM glucose (Fig. 1)
Effect of temperature
When preincubation was carried out at 48C with or without 500
mM glucose for 20 hr, ALA-D activity was not altered. A slight
variation (15%) between the enzyme activity evaluated without and
with glucose was detected when glycation was at 228C. Instead, at
378C, a great difference between ALA-D activity preincubated with
and without glucose was detected, reaching an inactivation of about
80% in the presence of the sugar. Hemoglobin glycation occurred
only at 378C under the assayed conditions (Fig. 2).
Effect of time
Preincubation with 500 mM glucose for different intervals showed
a sharp diminution of ALA-D activity already observed at the shortest time tested, reaching its lowest value after 20 hr of preincubation. Controls without sugar showed a diminution of enzyme activity, which was significantly lower than that detected in the presence
of the sugar (Fig. 3A).
GHb was determined in samples run in parallel, as a control of
FIGURE 2. Effect of preincubation temperature in the presence
of glucose on ALA-D activity (open bars) and GHb formation
(hatched bars). Red blood cells were preincubated for 20 hr at 4,
22 and 378C in the presence of 500 mM glucose. Results are expressed as percentage of the controls preincubated in the same
conditions without sugar at the indicated temperatures. Other experimental details were as indicated in the text. Control values
at zero time: ALA-D511.7760.70 U/mg Hb (n59); GHb5
0.6760.03 nmol HMF/mg Hb (n59). *P,0.05.
Reducing Sugars and ALA-D Inactivation
443
TABLE 1. Effect of different carbohydrates on ALA-D activity
Additions
Glucose
Galactose
Mannose
Rhamnose
Fructose
Xylose
Arabinose
Lactose
Maltose
Sorbitol
Glucosamine
2-Deoxyglucose
Enzymatic activity (%)
20.8
81.5
44.8
48.1
77.6
15.1
22.4
108.2
43.9
91.2
34.1
47.0
6
6
6
6
6
6
6
6
6
6
6
6
1.0*
3.3
2.1*
1.9*
3.9
0.9*
1.2*
5.4
2.6*
4.6
2.4*
2.8*
Red blood cells were preincubated 20 hr at 378C in the presence of the
indicated carbohydrates at a final concentration of 500mM. Results are
expressed as percentage of the controls preincubated without carbohydrate.
Other experimental details were as indicated in the legend to Figure 2 or
in the text.
*P , 0.05.
the glycation of hemoglobin (Fig. 4B), reaching the GHb basal levels
after 20 hr of preincubation in the presence of both agents.
DISCUSSION
FIGURE 3. Effect of preincubation time in the absence (h) or
presence (s) of glucose on (A) ALA-D activity, (B) GHb formation (C) and TBARS formation. Red blood cells were preincubated at 378C for 20 hr in all cases. Glucose (500 mM) was added
at different times during the whole period of preincubation, being
in contact with the enzymes at the indicated times (2, 4, 6, 14, 16
or 20 hr). Other experimental details were as indicated in the text.
*P,0.05.
Several studies in past decade have highlighted the importance of
hexose sugars, especially glucose, as being responsible for alterations
of cellular proteins and other biomolecules. It is well established
that nonenzymatic glycation alters the structural and the functional
integrity of a variety of enzymes (Brownlee, 1994). In this paper, we
provide evidence that, depending on the conditions of the glycation
changes in TBARS levels were detected along the whole period studied, either in the controls or in the glucose-treated RBCs (Fig. 3C).
Effect of other carbohydrates
With the aim of determining whether glycation was specific for glucose or whether it could also occur with other sugars, different
hexoses, pentoses and disaccharides were tested. All the hexoses assayed inactivated ALA-D between 50 and 80%, except galactose
and fructose, which reduced the enzyme activity by only 20%.
Pentoses (xylose and arabinose) strongly inactivated ALA-D
(80%). In glycation by disaccharides, lactose (a b-galactoside) did
not alter the activity of ALA-D; however, maltose (glucose homogeneous disaccharide) inhibited it by 60%. Sorbitol slightly reduced
ALA-D activity. Glucosamine reduced the enzyme activity by more
than 50% and 2-deoxyglucose produced a 50% inhibition (Table 1).
Effect of ASA
We relied on our in vitro glycation system to study the effects of ASA
on ALA-D inactivation and GHb formation. Preincubation with glucose (500 mM) and ASA (10 mM) prevented the inactivation of
ALA-D during the whole period studied (Fig. 4A) and even prevented
FIGURE 4. Effect of ASA on (A) ALA-D activity and (B) GHb
formation. Red blood cells were preincubated at 378C in the presence of glucose (500 mM) and ASA (10 mM) (d) or glucose (500
mM) (j) for different periods of time. Other experimental details
were as indicated in the text and in Figure 3. *P,0.05.
444
procedures, the activity of an enzyme of the heme pathway also can
be modified.
The dose–response curve for the inhibitory effect of glucose on this
enzyme was correlated with the glycation process, which was evaluated
through the formation of GHb. The level of GHb in blood has been
considered to indicate the degree of glycation of other proteins that are
freely exposed to circulating glucose (Schwartz, 1995).
Considering the effect of the temperature during glycation, we assume that this phenomenon would occur only at 378C, at least under our experimental protocol.
Although glycation appeared to be effective even at the shortest
periods assayed, its greater effects on ALA-D activity and hemoglobin glycation were detected at longer times.
Furthermore, the glycation procedure used here also proved to be
useful for investigating nonenzymatic glycation by sugars others
than glucose. Each hexose assayed inactivated ALA-D at a different
degree. It was demonstrated that 11 different sugars were able to
produce glycation of myofibrillar proteins and inactivation of ATPase activity (Syvory, 1994). Similar differences in the effects of
pentoses and disaccharides also were found.
From these findings, it appears that varying effects on the inactivation of the enzymes by different sugars could not be ascribed to
steric reasons but to differential affinity of the sugar for lysine groups
at or near the active site; the hydrophobicity of the active site environment also might play a significant role in the sugar–enzyme interaction (Brownlee, 1994).
Sorbitol did not inactivate ALA-D. It is interesting to recall that
Trüeb et al. (1980) reported that sorbitol, which does not bear a reactive aldehyde or keto group, only weakly binds collagen.
Beswick and Harding (1985) reported that the rate of binding of
2-deoxyglucose (a nonautoxidizable sugar) to lens a-crystallin is
similar to that of glucose (an autoxidizable sugar). These authors
concluded that it is the open-chain aldehyde of glucose that binds
initially to the protein amino groups and that there is no participation of dicarbonyl autoxidation products in the initial nonenzymatic
protein glycation reaction. In contrast, Wolff and Dean (1987) suggested that a component of protein glycation depends upon glucose
autoxidation and subsequent covalent attachment of ketoaldehydes.
It is noticeable that enzyme inactivation could result not only
from glycation, but also from the side effects of glucose autoxidation
involving the formation of free radicals, which might in turn indirectly act on enzyme activity (Jain and Palmer, 1997). In our system,
we could not attribute ALA-D inactivation to free-radical formation, because we did not detect any lipid peroxidation; however, accumulative effects occurring in vivo, can not be discarded, because
glycation itself could lead to formation of reactive oxygen species
(Yim et al., 1996).
Glycation of lens proteins might be a contributory factor in the
development of diabetic and senile cataracts. In vitro studies with rat
lens crystallins demonstrated that ASA inhibits cataratogenesis
(Cherian and Abraham, 1993).
Acetylation by aspirin would prevent glycation inhibition by
blocking the potential glycation residues (e-NH2 groups). Aspirin
inhibited in vitro and in vivo lens crystallin glycation, thiol oxidation
and the formation of high-molecular-weight aggregates (Swamy and
Abraham, 1989).
In our experimental model ASA was effective in inhibiting glycation of Hb as well as preventing ALA-D inactivation.
These results provide additional support for the potential use of
aspirin in the late stage of the Maillard reaction.
At present, investigations on the isolated enzyme are being per-
F. A. Caballero et al.
formed to identify the amino acid(s) taking part in the carbohydrate
linkage.
SUMMARY
The interaction between proteins and reducing sugars, known as the
Maillard reaction, proceeds through the formation of early products,
such as Schiff-base conjugates, to protein cross-linking, leading to
the alteration of their functions. A frequent coexistence of diabetes
and porphyria has been reported. The effect of in vitro glycation on
one of the heme pathway enzymes, (ALA-D), under several experimental conditions was investigated. In this paper, we provide evidence
showing that the activity of ALA-D can be modifided, depending on
the conditions of the glycation procedure. When preincubated with
500 mM glucose at 378C for 20 hr, ALA-D was 80% inactivated and
GHb levels were increased more than fourfold. ALA-D inactivation
could not be attributed to glucose autoxidation, because TBARS
were not modified during glycation. It has also been shown that
ASA (aspirin) was effective in preventing both Hb glycation and
ALA-D inactivation by glucose. These findings are providing further support for the potential use of aspirin during the late stages of
the Maillard reaction. A method has been developed for measuring
protein glycation in vitro, in a crude preparation of RBCs, that can
also be applied to sugars other than glucose.
E. Vazquez and A. M. del C. Batlle are members of the Career of Scientific Researchers at the National Research Council (CONICET). E. Gerez and F. Caballero are Research Assistants at the CONICET. This work was supported by
grants from the CONICET. A. M. del C. Batlle thanks the Ministerio de Educación y Ciencia, Spain, for special help. We are most grateful to Mrs. B. Corvalán
for technical assistance, and to Mr. M. Buzzi and Mr. Eng V. Di Massi, for buying some chemicals.
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