Research: Science and Education

www.JCE.DivCHED.org Vol. 84 No. 9 September 2007 Journal of Chemical Education 1541

Many, if not most, of the college students who take
chemistry courses are motivated by chemistrys importance
in biology and medicine. Effective teaching can take advan-
tage of such interests by relating chemical principles to medi-
cal practices. This helps to maintain student interest and
attention.
Diabetes has become increasingly prevalent in developed
countries in recent decades. The Centers for Disease Con-
trol estimates the U.S. diabetic population at over 20 mil-
lion, about 7% of the total population (1). The A
1c
blood
test is the principal means used to assess long-term control
of blood glucose concentration, and the CDC recommends
at least semiannual A
1c
blood testing for all diabetic patients.
The utility of this test derives from basic principles of chemi-
cal equilibrium and kinetics, and its results directly correlate
with complications of diabetes that arise from spontaneous
interactions of functional groups in carbohydrates and pro-
teins. The A
1c
test therefore provides useful and relevant il-
lustrations of general principles in introductory chemistry
courses, and the related chemistry provides interesting mate-
rial involving organic reactions.
Glucose
Glucose (blood sugar) provides a textbook example of
the famous Paracelsian principle that
All substances are poisons; there is none which is not a
poison. The right dose differentiates a poison from a
remedy. Paracelsus (14931541)
A healthy person maintains a blood glucose concentration
of 90 20 mgdL (5 1 mM) through feedback mecha-
nisms involving peptide hormones: insulin, which reduces
the concentration, and glucagon and epinephrine, which raise
it. If the concentration of glucose in blood falls below 50
mgdL, hypoglycemia, which can lead to coma and death,
results. On the other hand, diabetic patients suffer from hy-
perglycemia owing to insulin deficiency or insulin resistance.
The excess glucose is not immediately toxic, but its high con-
centration results in slow reactions in which it becomes
bonded to various proteins throughout the body (vide infra).
Affected organs include the blood vessels, leading to coro-
nary heart disease; eyes, leading to cataracts and retinopa-
thy; kidneys, leading to nephropathy; and nerves, leading to
neuropathy and limb necrosis (2).
Maintenance of a stable glucose concentration is a re-
markable feat, given that an average person processes about
160 grams of glucose per day, 120 grams in the brain alone.
About 20 grams of free glucose is available in body fluids at
a given time, and an additional 180200 grams is stored as
glycogen (polyglucose) in cells (3).
Instantaneous blood glucose concentrations can readily
be measured in a droplet of blood using ingenious devices
that rely on glucose oxidase-catalyzed oxidation of glucose
to gluconic acid and hydrogen peroxide,

followed by colorimetric determination of the peroxide con-
centration,
1

where HRP is horseradish peroxidase. However, the blood
glucose concentration changes rapidly and frequently, rising
upon food consumption and falling with physical activity,
and it would be necessary to monitor the instantaneous con-
centration almost continually to ascertain a meaningful av-
erage glucose concentration. This is clearly inconvenient and
impractical. Instead, the A
1c
test has been developed to pro-
vide a measure of blood glucose concentration averaged over
a period of several weeks. Maintaining a low average glucose
concentration over the long term is a high priority, as the
damaging effects of excess glucose, owing to its binding to
proteins (glycation), can be severe. The A
1c
test assays
glycation of a specific protein, hemoglobin (Hb), as a mea-
sure of overall protein glycation.
There are many clinical methods of measuring hemo-
globin glycation, which is reported as percent of total hemo-
globin bearing glycosyl groups. The methods vary in
specificity for the Hb A1
c
isomer. One group of methods (cat-
ion-exchange chromatography, agar gel electrophoresis) is
based on the reduced positive charge of the glycated protein,
and the other group (boronate affinity chromatography, im-
munoassay) is based on structural differences. The reference
method, used since 1978, is a HPLC cation exchange
method. The clinical goals and outcomes defined by the
19831993 Diabetes Control and Complications Trial were
based on the results from use of that reference method, and
The A
1c
Blood Test: An Illustration of Principles
from General and Organic Chemistry W
Robert C. Kerber
Department of Chemistry, SUNY at Stony Brook, Long Island, NY 11794-3400; rkerber@notes.cc.sunysb.edu
Products of Chemistry
edited by
George B. Kauffman
California State University
Fresno, CA 93740
Research: Science and Education
1542 Journal of Chemical Education Vol. 84 No. 9 September 2007 www.JCE.DivCHED.org
alternative methods are supposed to be calibrated to the same
scale. Recommended glycated hemoglobin levels are below
7%, with intervention recommended if they exceed 8% (2c).
The key point to be made in an introductory chemistry
course is that knowing that the rate of glycation is directly
dependent on the glucose concentration allows determina-
tion of an effective average concentration, despite wide short-
term variations. A high average glucose concentration is
deduced directly from detection of large quantities of glycated
hemoglobin. As pointed out by a referee, this dependence
can also be presented as an application of Le Chteliers prin-
ciple, as long as the rates are sufficient to ensure that the
glycation reactions are at equilibrium. From either the ki-
netic or the equilibrium perspective, the dependence of the
quantity of glycated hemoglobin on the average blood glu-
cose concentration should be evident, even in the absence of
detailed mechanistic modeling.
Hemoglobin
Hemoglobin is the protein, found in the red blood cells
(erythrocytes), that carries oxygen from the lungs to the tis-
sues of the body. Human hemoglobin is a tetrameric assem-
blage of four polypeptide chains, two designated ! and two
", each bound to a heme unit by coordination of a histidine
side chain with an ironheme moiety. Every milliliter of
blood has approximately 5 billion erythrocytes, and each
erythrocyte is packed with 280 million molecules of hemo-
globin (4). The concentration of hemoglobin molecules in
red blood cells is so high (340 mgmL, 2.3 mM) that they
almost could be said to be on the verge of crystallization.
The !
2
"
2
tetramers, spheroids of axial dimensions 65 by 55
by 50 , are only 10 apart on the average (5).
Also at relatively high concentration within the eryth-
rocyte is glucose, whose transport across the cell membrane
is facilitated by transporter proteins. This maintains the in-
tracellular glucose concentration close to that in the serum
outside (6). Consequently, the erythrocyte interior provides
a space where glucose and a specific protein, hemoglobin,
come together in high concentrations, a favorable condition
for a bimolecular reaction.
Early studies of hemoglobins by ion-exchange chroma-
tography had revealed ubiquitous minor components with
reduced positive charges relative to unmodified hemoglobin.
The principal component that met these criteria was desig-
nated hemoglobin A
1c
(7). This component constituted 5
7% of the total hemoglobins in normal patients, but the
quantity rose as high as 20% in diabetic patients. Hemoglo-
bin A
1c
was eventually identified as a product of spontane-
ous reaction of normal hemoglobin with glucose (2).
Kinetics and Mechanism of Glycation
Glycation provides an example of a biologically impor-
tant reaction that is not enzyme-catalyzed. Initial reaction of
glucose and hemoglobin involves reversible formation of an
imine:

RCH(OH)CH N(Hb) + H
2
O
RCH(OH)CHO + (Hb)NH
2

k
+1
k
#1
(1)
This is followed by a less reversible, exergonic tautomeriza-
tion of the imine to an aminoketone (a deoxyfructose de-
rivative), often referred to as an Amadori rearrangement:

k
+2
k
#2
RC( O)CH
2
NH
2
(Hb)
+
H
+
+ RCH(OH)CH N(Hb)
(2)
The kinetics of hemoglobin glycation has been studied
both in vitro and in vivo (810). Results are complicated by
the simultaneous reaction of the glucose at several of the ac-
cessible amine side chains in hemoglobin (there are eleven
lysines and one N-terminal valine on each of the four chains
of the !
2
"
2
tetramer). Fortunately, a set of parallel second-
order reactions between a given pair of reactants shows over-
all second-order behavior that can be described by a
composite rate constant. Individual rate constants (if needed)
can then be determined from product ratios (11). The over-
all rate of reaction is also affected by various ligands that bind
to hemoglobin, including phosphate ions (12), organic phos-
phates (13), and oxygen (13a, 14).
Erythrocytes have an average lifespan of about 120 days
in the body, so the occurrence of glycation reactions leads to
a steady-state concentration of glycated hemoglobins. Their
concentration is directly dependent on the average glucose
concentration over the weeks prior to the determination. This
averaging property is the basis for the usefulness of the A
1c
test as a measure of effectiveness of glucose control.
Reported values for the rate and equilibrium constants
for the steps in reactions 1 and 2 under physiological condi-
tions vary widely, but the use of consensus values (k
+1
= 96
10
6
L mol
1
s
1
; k
1
= 100 10
6
s
1
; k
+2
= 14.2 10
6
s
1
;
k
2
= 1.7 10
6
s
1
) has led to a biokinetic model that agrees
with clinical data relating average glucose concentration to
hemoglobin A
1c
(10). This set of rate constants may provide
an interesting example for steady-state or numerical model-
ing in a physical chemistry course.
2
A sample calculation is
presented in the Supplemental Material.
W
The second-order kinetics that has been experimentally
verified for glycation of hemoglobin is assumed to apply to
other proteins as well. A high result on the A
1c
test indicates
a high average concentration of glucose during the weeks pre-
vious to the test and therefore a proportionately high level
of glycation of other proteins. Indeed, proteins with a longer
residence time in the body than hemoglobin would undergo
continuous glycation, rather than achieve the steady state that
results from the regular turnover of hemoglobin. It is the
glycation products from other proteins that are responsible
for the toxic effects of excess glucose; the glycated hemoglo-
bin that is measured in the test serves as a convenient indi-
cator of the ongoing extent of glycation and therefore an
indication of glucose toxicity.
Structures of Glycation Products
The N-terminal valines of the two hemoglobin "-chains
are generally the most reactive glycation sites in vivo, and
the hemoglobin A
1c
designation refers specifically to the stable
product of eq 2 at these two sites. The enhanced reactivity
of these sites relative to the other 46 primary amino groups
of the hemoglobin tetramer probably results from their greater
Research: Science and Education
www.JCE.DivCHED.org Vol. 84 No. 9 September 2007 Journal of Chemical Education 1543
accessibility and weak basicity [pK
a
of 6.8 (13a, 15) as com-
pared to about 10.5 for a lysine side chain], which leaves them
partly unprotonated and nucleophilic at physiological pH.
It has also been argued that the histidine residue adjacent to
the N-terminus of the "-chain enhances its overall reactivity
by catalyzing the Amadori rearrangement (13b, 14).
Less prevalent glycated hemoglobins having carbohydrates
other than glucose attached to the "-chain N-terminus have
been identified (16), as have isomers of hemoglobin A
1c
with
glucose attached to the ! N-terminus and to lysine residues
on either chain (17). Phosphate or organophosphate ions di-
rect the site of glycation in favor of the preferred "-terminal
valine, an effect attributed to their enhancement of the rate
of the Amadori rearrangement (12, 13b). Intramolecular ca-
talysis of Amadori rearrangement by adjacent carboxylate
groups has similarly been cited in one rationalization of the
enhanced reactivity of certain specific lysine residues (17b, 18).
Recent advances in protein analysis by electrospray and
MALDI mass spectrometry promise to accelerate the analy-
sis of glycated hemoglobins (19). These methods of analysis
show hemoglobins !-chain to be glycated to about two-thirds
the extent of the "-chain, rather more than suggested by clas-
sical methods of analysis (20). Polyglycation of the "-chains
is also evident at higher glucose concentrations (21).
Advanced Glycation End Products, AGEs
The glycated hemoglobin A
1c
measured in the test is not
toxic. But the result is a surrogate for analogous spontane-
ous glycations involving other proteins throughout the body.
The intermediate glycation products analogous to the
deoxyfructosyl-lysine shown in eq 2 undergo additional slow
non-enzymatic reactions, collectively referred to as Maillard
reactions that result in functional degradation of the proteins.
The complex products of these slow reactions are referred to
as advanced glycation end products, AGEs (22). Along with
the mechanisms underlying eqs 1 and 2, the chemistry un-
derlying formation of these AGEs provides many group work-
shop problems useful in organic chemistry courses.
One such reaction is the oxidation of the deoxyfructosyl-
lysine to carboxymethyl-lysine and erythronic acid (23),

(3)
catalyzed by phosphate and inhibited by metal-chelating
agents; a free-radical mechanism occurring via the enediol
has been suggested (23). The carboxymethyl products are not
particularly toxic, and their formation may actually limit the
competitive formation of more damaging byproducts (23).
Scheme I. Formation of glyoxal imine intermediates.
Research: Science and Education
1544 Journal of Chemical Education Vol. 84 No. 9 September 2007 www.JCE.DivCHED.org
These more toxic products are the result of formation
of highly reactive !-dicarbonyl compounds (!-oxoaldehydes)
or imines by a sequence of enolizations, dehydrations, and
retro-aldol reactions. These reactions occur slowly in glucose
solutions under physiological conditions, but more rapidly
in the presence of N
!
-protected lysine or proteins capable of
imine formation (24). Formation of glyoxal imine interme-
diates is illustrated in Scheme I. These imines may undergo
hydrolysis to glyoxals or they may condense directly with
lysine residues from proteins via transimination reactions to
form the various toxic products described below. These al-
ternatives are not explicitly shown in Scheme I to reduce com-
plexity.
The highly electrophilic !-oxoaldehydes and imines are
the direct precursors to the AGEs (25, 26). They react with
particular facility with the arginine residues of proteins to
form heterocyclic products, including imidazolones and py-
rimidines (Scheme II) (26, 27). Since arginine residues in
proteins have a high probability of occurrence in ligand and
substrate recognition sites and enzyme active sites, these un-
controlled derivatization reactions often lead to functional
disruption (25).
Also highly damaging are reactions that result in inad-
vertent cross-linking of protein chains, which reduces their
ability to carry out normal functions and contributes to cir-
culation, joint, and vision problems in diabetics and the aged.
One such cross-link is produced by reaction of two lysine
residues from proximate chains with !-oxoaldehydes to form
a bis(lysyl)imidazolium cross-link (Scheme III) (27, 28).
The pentosidine cross-link,
similarly forms a stabilized aromatic heterocycle linking two
protein chains, in this case through an arginine residue on
one and a lysine on the other. The five-carbon moiety that
makes up the rest of the heterocycle derives from a pentose,
probably ribose (29).
These protein-altering reactions occur spontaneously,
without enzyme catalysis. The products result in reduction
of protein activity and flexibility and hence to cell damage.
These altered proteins are found in everyone, and their quan-
tity increases with age. But uncontrolled diabetics have un-
usually high quantities of circulating glucose, so the quantities
of AGEs formed in their bodies are significantly higher than
in non-diabetics of the same age. (In this sense, diabetics age
faster.)
Scheme II. Formation of heterocyclic AGEs.
Scheme III. Formation of bis(lysyl)imidazolium cross-links.
Research: Science and Education
www.JCE.DivCHED.org Vol. 84 No. 9 September 2007 Journal of Chemical Education 1545
Conclusion
The hemoglobin A
1c
test provides a convenient measure
of long-term average glucose concentration, which indicates
the extent of ongoing glycation of proteins in the body. For-
mation of the glycation products responsible for many of the
debilitating effects of diabetes is directly proportional to the
average glucose concentration in the bloodstream, which can
be viewed either as a consequence of their second-order re-
action (first order each in glucose and in protein) or of Le
Chteliers principle applied to the equilibria of eqs 1 and 2.
The enhanced occurrence of these spontaneous reactions
in uncontrolled diabetics can be used by teachers in intro-
ductory courses to illustrate the consequences of kinetic or-
der. The spontaneous nature of these reactions makes them
particularly straightforward examples of the effect of concen-
tration on rate in medically relevant reactions. Detailed ki-
netic analyses may be carried out in advanced physical
chemistry courses.
3
The glycation reactions provide interest-
ing examples whose mechanisms involve sequences of simple
steps (e.g., imine formation, tautomerization, condensations)
that can be worked out by team-learning groups in second-
semester organic chemistry courses. The biological and medi-
cal relevance of the reactions should provide immediacy and
motivation to the students.
W
Supplemental Material
This set of rate constants may provide an interesting ex-
ample for steady-state or numerical modeling in a physical
chemistry course. A sample calculation is presented in this
issue of JCE Online.
Notes
1. Use of a related glucometer in an error analysis laboratory
exercise has been suggested by Edmiston, P. L.; Williams, T. R. An
Analytical Experiment in Error Analysis: Repeated Determination
of Glucose using Commercial Glucometers. J. Chem. Educ. 2002,
77, 377379.
2. A referee reports that he found it a somewhat daunting,
but ultimately rewarding challenge to build a spreadsheet to pre-
dict the time variance (of glycated and unglycated hemoglobins),
based on the rate constants provided...
3. Adapted from ref 22.
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