Clinica Chimica Acta 277 (1998) 159–170

Comparison of advanced glycation endproducts on

haemoglobin (Hb-AGE) and haemoglobin A 1c for the
assessment of diabetic control

Z. Turk a , *, R. Mesic´ a , B. Benko b

a
University Clinic for Diabetes, Endocrinology and Metabolic Diseases, Dugi dol 4 A, HR-10000 Zagreb,
Croatia
b
Institute of Immunology, Zagreb, Croatia

Received 4 February 1998; received in revised form 27 July 1998; accepted 7 August 1998

Abstract

Glycation process in vivo results in two different products: early and advanced glycation
endproducts (AGEs). The mechanism of early product formation has been well described, with
HbA 1c as the best-studied example. The finding that advanced glycation endproducts are also
formed on haemoglobin suggests that HbA 1c is a precursor for Hb-AGE formation. HbA 1c has
been well established as an important indicator for glycaemia monitoring, but the diagnostic role
of Hb-AGE has not yet been clarified. A question is whether HbA 1c and Hb-AGE are competitive
or complementary parameters. In our study, Hb-AGE was quantified by the competitive ELISA
technique using polyclonal anti-AGE–RNase antibodies to detect AGE immunoreactivities of
proteins precipitated in red cell hemolysate. Results are expressed as AGE units / mg Hb. Hb-AGE
was analysed in three groups of patients divided according to HbA 1c values as follows: group I
(n 5 25) HbA 1c , 7%, Hb-AGE 5 6.93 (5.7–7.3) U / mg; group II (n 5 25) HbA 1c 5 7–10%,
Hb-AGE 5 8.62 (7.7–10.2) U / mg; and group III (n 5 25) HbA 1c . 10%, Hb-AGE 5 12.47
(10.8–13.9) U / mg (median (interquartile range)). A close relation between the amounts of red cell
HbA 1c and Hb-AGE was observed in all diabetic subjects (n 5 75) r 5 0.77, P , 0.001. Patients
with HbA 1c level . 8% were considered to be in poor glycaemic control and those with
HbA 1c , 8% in good control. In the well-controlled subgroup (n 5 33), HbA 1c and Hb-AGE were
less tightly correlated (r 5 0.37, P , 0.001). However, in those patients with a higher level of
HbA 1c 5 12.55 (8.9–13.3)% (n 5 42), the related Hb-AGE was 11.5 (10.3–12.8) U / mg Hb,
yielding a more significant correlation (r 5 0.51, P , 0.001). The content of Hb-AGE did not
correlate with age (r 5 0.09), diabetes duration (r 5 0.05) or severity of retinopathy and / or
nephropathy. The observed difference may reflect a different kinetic rate of HbA 1c production and

*Corresponding author. Tel.: 1385-1-2332222; fax: 1385-1-2331515; e-mail: zturk@indija.idb.hr

0009-8981 / 98 / $ – see front matter  1998 Elsevier Science B.V. All rights reserved.
PII: S0009-8981( 98 )00128-4
160 Z. Turk et al. / Clinica Chimica Acta 277 (1998) 159 – 170

subsequently the rate of Hb-AGE formation. The discrepancy in the correlation between HbA 1c
and Hb-AGE suggests that they are complementary rather than opposed parameters. The amount
of haemoglobin-linked AGEs does not correlate with the presence or absence of retinopathy
and / or nephropathy. It seems that Hb-AGE represents only the metabolic status, equally in the
subjects with and without diabetic microangiopathy.  1998 Elsevier Science B.V. All rights
reserved.

Keywords: Diabetes mellitus; Advanced glycation endproducts; Glycated haemoglobin

1. Introduction

Nonenzymatic glycation of macromolecules is recognised as an important

biogenetic process [1]. In vivo glycation results in two different products: early
(EGP) and advanced glycation endproduct (AGE). In the early step of the
glycation pathway, reversible N-glucosylamine (Schiff base) forms and reaches
equilibrium over a period of several hours. Then the N-glucosylamine undergoes
a characteristic intramolecular rearrangement reaction to form chemically
stabilised 1-amino-1-deoxyketose or Amadori product [2]. The chemistry of
early glycation products has been well described, with HbA 1c as the best-studied
example [3]. Of great significance is the fact that these early glycation products
are direct, chemical precursors of a group of more slowly forming advanced
endproducts. A protein with early product can be cross-linked through its
reactive carbonyl group to other proteins, lipoproteins or other amine-containing
substrates, forming a variety of advanced glycation endproducts. Advanced
glycation compounds arise by the slow rearrangement, dehydration, and
condensation of early products to produce irreversibly bound moieties that
persist for the lifetime of the protein [4]. Advanced glycation endproducts are
known for their heterogeneity, and the precise chemical structure of the majority
of AGE compounds still remains to be elucidated. However, some chemical
structures have been identified, among them: N-carboxymethyl-lysine, pen-
tosidine, pyrraline, N-lactatolysine, and imidazolone derivates [5]. Through
AGE effects on the functional properties of extracellular matrix, intracellular
signal transduction, and protein function, AGEs probably have a central role in
the pathogenesis of diabetic complications [6]. A concern in the study of AGE
biochemistry has been the fact that AGE is an immunogen and that it produces
antiserum [7,8]. Enzyme-linked immunosorbent assay (ELISA) using AGE-
specific antibodies proved a useful parameter in measuring AGE formation as
well as in localising AGE within specific tissues.
The finding that AGEs are also formed on haemoglobin suggests that HbA 1c
is a precursor of chemically irreversible Hb-AGE formation [9]. HbA 1c has been
 Z. Turk et al. / Clinica Chimica Acta 277 (1998) 159 – 170 161

well established as an important indicator for glycaemia monitoring, but the

diagnostic role of Hb-AGE has not yet been clarified. Here we investigate
whether HbA 1c and Hb-AGE are competitive or complementary parameters. In
order to evaluate these parameters, we first developed and standardise an ELISA
method for qualitative and quantitative follow up of Hb-AGE.

2. Material and methods

2.1. Material

Bovine pancreatic ribonuclease A (RNase A), bovine serum albumin, rabbit

albumin, and anti-rabbit IgG alkaline phosphatase conjugate were purchased
from Sigma (USA). Sephacryl S-200 HR was obtained from Pharmacia
(Uppsala, Sweden); human serum albumin (Cohn fraction V) from the Institute
for Immunology, Zagreb, HR; D-glucose and all other chemical were from
Merck (Darmstadt, Germany).

2.2. Preparation of AGE–proteins

AGE–RNase was prepared by incubating RNase (25 g / l) with 1.0 mol / l

D-glucose in 0.2 mol / l sodium phosphate buffer, pH 7.4, for up to 90 days. The
reaction mixture was sterilised by filtration (0.22-mm pore filter) and allowed to
incubate in the dark at 378C. AGE–proteins derived from human albumin and
from e-amino-octanoic acid were prepared by glycation in vitro in a 0.5 mol / l
sodium phosphate buffer. The kinetics of albumin glycation (50 g / l) for a period
of between 7 and 90 days at different solution acidities (pH 4.5–8.0),
temperature of 378C and D-( 1 )-glucose concentration (54 mmol / l), was
followed by spectrophotometry. Isoelectric focusing was used in the analysis of
glycated albumin (Phastsystem, Pharmacia, Sweden). Haemoglobin-AGE was
prepared by incubating 159 g / l of haemoglobin with 30 mmol / l of glucose in
sodium phosphate buffer, pH 7.4, for 75 days at a temperature of 378C. The
reaction mixture was sterilised by filtration.
Amadori compound, i.e. 1-deoxy-1-morpholino-D-fructose (DMF), was ob-
tained from D-glucose and morpholine on heating in alcoholic solution, followed
by the addition of ethyl malonate, yielding a fructose derivate as described
elsewhere [10].

2.3. Immunisation

Multiple standard rabbit (New Zealand strain) immunisation by AGE–RNase

(eight times, every 14 days, a dosage of 2 mg / kg weight, first time with
162 Z. Turk et al. / Clinica Chimica Acta 277 (1998) 159 – 170

complete and subsequently with incomplete Freund adjuvant) was used in the
preparation of advanced glycation endproduct-specific antiserum. Direct and
competitive ELISA tests determined the cross-reactivity of antiserum with
AGE–albumin and AGE–e-amino octanoate, and absence of major non-specific
reactions.

2.4. Haemoglobin sample preparation and HbA1 c measurement

Samples were prepared according to Jeppsson et al. [11]: heparinised blood

(600 ml) was incubated for 4 h at 378C with saline and centrifuged. The
supernatant was discarded and the erythrocyte pellet was hemolysed by the
addition of H 2 O and CCl 4 . Separation of the cell ghosts was accomplished by
centrifugation, the clear supernatant was diluted with malonic buffer and
analysed. HbA 1c was separated from other haemoglobins by cation-exchange
chromatography (fast protein liquid chromatography system, Mono S HR 5 / 5
column at pH 5.7) and expressed as percentage of the total amount of
haemoglobin. The hemolysate was further used for Hb-AGE determination and,
for that purpose, it was delipidated with toluene. Haemoglobin was precipitated
by ice-cold 30% trichloroacetic acid and the sedimented protein was dissolved in
NaOH. The pH of this solution was adjusted to 7.4 by dilution buffer containing
0.2 mol / l sodium phosphate with 60 mg / l urea, 1.0 g / l sodium dodecylsulphate
and 0.5 g / l Triton X-100. Hb-AGE was measured by the ELISA technique and
hemolysate was diluted in a range of 1:100 to 1:2000. The Hb-protein
concentration was determined according to Lowry et al. [12].

2.5. Immunoassay

The enzyme-linked immunosorbent assay (ELISA) was performed in two

different modes, i.e. as a direct and a competitive ELISA. The wells of
immunoplates (Biohit high binding microwell strip plate) were coated with
AGE–human serum albumin, 0.5 mg / well in 0.05 mol / l carbonate buffer, pH
9.6, incubated for 3 h at room temperature and overnight at 48C. The wells were
washed three times with 0.3 ml washing buffer (saline containing 0.5 ml / l
Tween-20 and 1 mmol / l NaN 3 ). The wells were then blocked by incubating for
12 h with 0.3 ml of carbonate buffer 0.05 mol / l, pH 9.6, containing 5 g / l
gelatine. After washing, the wells were additionally blocked by glycine in 0.05
mol / l Tris, pH 8.8, for further 12 h. Washing was performed first with 0.3 mol / l
NaCl and then with washing buffer.
A non-competitive ELISA determined antiserum titers. Rabbit antiserum was
diluted with PBS containing 0.5 ml / l Tween-20 plus 10 g / l BSA and 0.1 ml of
dilutions ranging from 1:500 to 1:8000 were added to the wells, incubated for 3
h at room temperature and overnight at 48C. After incubation and washing, the
 Z. Turk et al. / Clinica Chimica Acta 277 (1998) 159 – 170 163

wells were treated with another antibody labelled with the enzyme. To each
well, 0.1 ml of alkaline phosphatase-linked anti-rabbit IgG from sheep diluted
with PBS containing 10 g / l BSA was added. Following incubation for 1 h at
378C, the plate was extensively washed and developed utilising p-nitrophenyl
phosphate (16 mmol / l) as a colorimetric substrate in 0.4 mol / l 2-amino-2-
methyl-propanol buffer. After a convenient time, an alkali stopping solution was
added and the degree of enzymatic turnover of substrate was determined by dual
wavelength absorbance measurement at 405 and 630 nm by an ELISA reader.
For the performance of a competitive ELISA with polyclonal antibodies, one
half of the immunoplate was coated by adding 0.1 ml AGE–human serum
albumin, 0.5 mg / well in 0.05 mol / l carbonate buffer, pH 9.6, while the other
half of the plate served for the detection of non-specific binding. All wells were
blocked in two steps, first by 5 g / l gelatine and then by glycine, as previously
described. Thereafter, 0.05 ml of Hb-AGE or hemolysate (i.e. competing
antigen) diluted with PBS–Tween-20, and 0.05 ml of anti-AGE–RNase an-
tiserum (dilution 1:1000) was added to the wells. Competitors were added to the
wells at concentrations ranging from 10 23 to 10 3 mg / well. The plate was
incubated for 3 h at room temperature and overnight at 48C. After incubation,
the plate was rinsed first with 0.3 mol / l NaCl and then with washing buffer, and
the wells were treated with other antibodies labelled with the enzyme, as
described for the non-competitive ELISA. The difference between sample
absorbance and blank absorbance was calculated. Results were expressed as
B /B0 , where B was competitor absorbance, B0 absorbance in the absence of
competitor along with correction for non-specific binding (NSB), and the
calculation followed the relationship: B /B0 5 (B 2 NSB):(B0 2 NSB). A cali-
bration curve was plotted from serial double dilutions of AGE–human serum
albumin, corresponding to protein concentrations of 100–3.12 mg / l. Competi-
tive immunoreactivity of the samples was read from the calibration curve and
expressed relative to AGE–albumin standard in AGE unit / mg Hb. Each
hemolysate was analysed in three dilutions, which were applied in quadruplicate
on the plate. The intra-assay variation for this procedure was 7.6% and the
inter-assay variation was 9.8%.

2.6. Patients

Seventy-five diabetic patients (IDDM and NIDDM) were enrolled in the

study. There were 39 female and 36 male subjects. Patients’ ages ranged from
27 to 54 years, mean age 39 years. Diabetes duration was from 3 to 25 years
with the mean of 11.7. Thirty-eight patients had clinically established re-
tinopathy and / or nephropathy. Retinopathy was assessed by an ophthalmologist,
whereas nephropathy was defined by obligatory proteinuria ( . 0.5 g urinary
protein / 24 h), with decreased creatinine clearance. The studied subjects were
164 Z. Turk et al. / Clinica Chimica Acta 277 (1998) 159 – 170

diabetic patients treated at the clinic for diabetes. Samples for Hb-AGE
measurements were taken during routine control of glycaemia. The Ethics
Committee and Expert Council of the Clinic for Diabetes, Endocrinology &
Diseases of Metabolism in Zagreb had approved the study.

2.7. Statistical analysis

Concerning asymmetric distribution of variables, results are expressed as

median (interquartile range). Wilcoxon matched-pair test and Spearman correla-
tion were used for the analysis of obtained data. The limit of significance was
defined as P , 0.05.

3. Results

Advanced glycation albumin was prepared by the in vitro incubation of

bovine albumin and human albumin with glucose as heterologous carrier
proteins for the AGE epitope. Albumin glycation yielded a chromophore, which
absorbed light in the visible and ultraviolet regions. All AGE-modified proteins
showed absorption and fluorescent spectra. The change of colour is a function of
time, temperature, start of dilution acidity, and concentrations of protein and
sugar. The dilution in which the reaction occurs gains acidity in parallel to the
change of colour. Glycated albumin gel chromatography revealed a considerable
formation of albumin dimmers and polymers (Fig. 1). Isoelectric focusing
showed major changes in the isoelectric point of glycated albumin, which leads
to heterogeneity of albumin molecules.
Antibodies against advanced glycated RNase were prepared essentially
according to Makita et al. [8]. A direct ELISA technique showed that antiserum
from rabbits immunised with advanced glycated RNase reacted with AGE–
human albumin, AGE–bovine albumin, AGE–haemoglobin, AGE–e-amino-
octanoate and AGE–RNase, but not with unmodified albumins, haemoglobin or
RNase, suggesting the presence of antibodies specific for the AGE modification.
Competitive ELISA tested the specificity of the anti-AGE antiserum with
AGE-modified and unmodified proteins as competing antigens. AGE modi-
fication was observed to compete for antibody binding when it is present on
diverse carrier proteins. Thus, AGE–HSA, AGE–Hb, AGE–BSA, AGE–e-
amino-octanoate and AGE–RNase all demonstrate specific inhibition of anti-
body binding to glucose modified AGE–human albumin. At the same time,
unmodified HSA, Hb, BSA, e-amino-octanoate and RNase did not compete in
this ELISA test. The Amadori product, i.e. deoxymorpholinofructose, also failed
to inhibit antiserum binding. The inhibition curves of different AGE–proteins
were parallel to each other. Fig. 2 shows an ELISA competition curve for
 Z. Turk et al. / Clinica Chimica Acta 277 (1998) 159 – 170 165

Fig. 1. Chromatogram of glycated human albumin (2 ml, 50 g / l), Sephacryl S-200 HR, 2.5395
cm, 0.05 mol / l phosphate buffer, pH 7.0, flow rate 20 ml / h, recorded at three wavelengths. The
absorption at 350 nm is mostly caused by AGE chromophores, while AGE chromophores and the
protein itself induce the absorptions at 300 and 280 nm. The first peak is characteristic of albumin
polymers and dimmers, while the second one corresponds to albumin monomer.

anti-advanced glycation RNase antiserum. The calibration curve was plotted

from serial dilutions of AGE–human serum albumin. According to the in-
dependent kinetic measurement (results not shown), the reaction of alkaline
phosphatase with p-nitrophenyl phosphate under the chosen conditions is a
linear function of time for at least 60 min. The non-linear least-squares fit
procedure of the experimental points to the empirical function of the type
B 12a
] 5 ]]]n 1 a
B0 1 1 bC
is used for the construction of calibration curve. C is the concentration or the
amount of inhibitor, and a, b and n are the fitting parameters (a50.0657;
b50.3354; n50.9607).
The competitive ELISA technique was used to detect AGE immuno-
reactivities of haemoglobin in red cell hemolysate. Haemoglobin A 1c was
measured by means of HPLC in the same samples. Haemoglobin-AGE level was
determined in 75 diabetic patients with different glycaemic control. Haemo-
globin A 1c values in the observed patient group ranged from 5.0 to 15.2%, and
the patients were divided into three groups according to their level of glycated
haemoglobin. The first group consisted of 25 individuals with HbA 1c values
,7% and median Hb-AGE value 6.93 (5.7–7.3) U / mg Hb. The second group
166 Z. Turk et al. / Clinica Chimica Acta 277 (1998) 159 – 170

Fig. 2. Competitive inhibition of AGE-specific antibodies by AGE–human serum albumin.

AGE–HSA was prepared by the incubation of purified human albumin (50 g / l) at 378C in 0.5
mol / l phosphate buffer, pH 7.5, containing 54 mmol / l D-(1)-glucose. The competitive ELISA
was performed as precisely described in Section 2. The curve drawn through the experimental
points was constructed using non-linear least-squares fit procedure and formula B /B0 5(12a) /
(11bC n)1a, where C is the amount of albumin added into the well, and a50.0657, b50.3354
and n50.9607 are the fitting parameters.

(n525) had HbA 1c levels within a range of 7–10% and the related median
Hb-AGE was 8.62 (7.7–10.2) U / mg Hb. The highest values of Hb-AGE (12.47
(10.8–13.9) U / mg Hb) were found in 25 patients with HbA 1c .10%. When
these data are considered in terms of percentage, the level of Hb-AGE exceeded
HbA 1c by 12 and 2% in the first and second groups, respectively, whereas in the
third group the level of Hb-AGE was lower by 5.6% than the level of HbA 1c
(Fig. 3). A close relation between the amounts of red cell HbA 1c and Hb-AGE
was observed in all diabetic subjects (n575): y52.410.77x; r50.77 P,0.001
(Fig. 4). However, the analysis of correlation between these two parameters
according to glycaemic control was more interesting. Namely, diabetic patients
with a HbA 1c level .8% were considered to be in fair-to-poor glycaemic
control and those with HbA 1c ,8% in good control. In the well-controlled
subgroup (n533), HbA 1c 56.1 (5.4–7.5)% and Hb-AGE57.26 (6.1–8.0) U / mg
Hb, were less tightly correlated (r50.37, P,0.001). However, in those patients
with a median HbA 1c 512.55 (8.9–13.3)% (n542), the related Hb-AGE was
11.5 (10.3–12.8) U / mg Hb, yielding a more significant correlation (r50.51,
P,0.001). The Hb-AGE did not correlate with age (r50.09), diabetes duration
(r50.05) or severity of retinopathy and / or nephropathy.
 Z. Turk et al. / Clinica Chimica Acta 277 (1998) 159 – 170 167

Fig. 3. The course of advanced glycation endproducts on haemoglobin (Hb-AGE) and glycated
haemoglobin A 1c in 75 diabetic patients divided according to glycaemic control: group (I)
HbA 1c ,7%; group (II) HbA 1c 57–10%; group (III) HbA 1c .10%. The points represent the
median values in 25 subjects. Hb-AGE is expressed in units of AGE per mg haemoglobin, and
HbA 1c as percentage of the total amount of haemoglobin.

4. Discussion

Advanced glycation endproducts were prepared in vitro by incubation of

glucose with a variety of proteins. We observed that AGE formation depended
considerably on dilution acidity. Since the pH range in different tissues of the
body is rather wide, the glycation process may be expected to develop faster
when accompanied by higher pH. Previous studies have shown that AGE-
modified keyhole limpet haemocyanin [14], AGE–albumin [7] or AGE–RNase
[8] can be used as immunogens to prepare antiserum specific for a common
AGE epitope that forms in vitro and in vivo [15]. An antibody of comparable
specificity was prepared in our laboratory and used for Hb-AGE measurement.
AGE antibodies recognise AGE epitopes whose nature is still unknown. It was
because of the heterogeneity of AGE structures that we used polyclonal rather
168 Z. Turk et al. / Clinica Chimica Acta 277 (1998) 159 – 170

Fig. 4. Correlation between HbA 1c as an early glycation product and amounts of advanced
glycation endproducts on haemoglobin (Hb-AGE) in a group of 75 diabetic patients with different
glycaemic control ( y52.410.77x; correlation coefficient r50.77; P,0.001). HbA 1c was
measured by high-performance liquid chromatography, and Hb-AGE by competitive ELISA.

than monoclonal anti-AGE antibodies in our study. Competition ELISA studies

showed the cross-reactive AGE epitopes to form on a variety of carrier proteins,
including in vitro glucose-derived albumins, haemoglobin or RNase, as well as
AGEs on serum proteins or haemoglobin glycated under physiological con-
ditions.
In the ELISA method haemoglobin incubated long-term with glucose was
used as a competitive antigen. However, it showed a surprisingly weak
inhibition. In a repeated trial, the same haemoglobin sample had previously
undergone TCA–NaOH treatment according to Makita et al. [13]. This
haemoglobin sample had an inhibition effect in a competitive ELISA test. A
possible explanation might be that some AGE modifications remain unattainable
to antibodies due to the steric configuration of the haemoprotein molecule.
However, it is possible that protein denaturation occurs during TCA–NaOH
 Z. Turk et al. / Clinica Chimica Acta 277 (1998) 159 – 170 169

treatment, making the formerly hidden AGE epitopes accessible to the anti-
bodies.
The diagnostic value of Hb-AGE measurement was evaluated in diabetic
patients with different glycaemic control. A better correlation between the
HbA 1c level and amount of Hb-AGE was observed in patients with fair-to-poor
metabolic control, whereas in patients with HbA 1c ,8% the same parameters
had a much weaker correlation. The observed difference may reflect a different
kinetic rate of HbA 1c production and subsequently the rate of Hb-AGE
formation. A precise temporal relation between the formation of early and
advanced glycation parameters has not been established. At the moment it is
known that Hb-AGE declines more slowly than HbA 1c . The time-dependent
decline in mean circulating Hb-AGE levels is slower by 23% in comparison
with HbA 1c [16]. However, one of our results is controversial to the cited data.
As shown in Fig. 3, the level of Hb-AGE exceeded the level of HbA 1c in groups
(I) and (II), whereas in group (III) (HbA 1c .10%) the mean level of Hb-AGE
was lower than that of HbA 1c . This led us to speculate on the possibility that red
cells undergo intensive modification by advanced glycation and faster elimina-
tion from the circulation via a specific receptor system [17]. It is also possible
that the differences we observed in this study were partly due to possible
susceptibility of circulating red cell haemoglobin to modification by glycotoxins,
reactive plasma AGE fragments that occur in patients with impaired renal
function [9].
The discrepancy in the correlation between HbA 1c and Hb-AGE suggested
them to be complementary rather than opposed parameters. It means that
Hb-AGE level should not be considered a substitute for the traditional diagnostic
tool of HbA 1c but rather its supplementation. The amount of haemoglobin-linked
AGEs do not correlate with the presence or absence of retinopathy and / or
nephropathy. It seems that Hb-AGE represents only the metabolic status, equally
in the subjects with and without diabetic microangiopathy.

Acknowledgements

We are indebted to Mrs Barbara Kos for her excellent technical assistance in
immunisation of rabbits. The Ministry of Science and Technology of the
Republic of Croatia supported this study.

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