Clinica Chimica Acta, 136 (1984) 75-81 75

Elsevier

CCA 02729

Glucosylation of human haemoglobin A. Dynamic

variation in HbA,, described by a biokinetic model
Henrik B. Mortensen a,*, Aage Verlund b and Carsten Christophersen ’
a The Pediatric Department, Glostrup Hospital, b Nouo Research Institute and the ’ Marine Chemistry
Section, Department of General and Organic Chemistry, The H. C. @wed Institute, University of Copenhagen
(Denmark)

(Received July 9th, 1983)

Key words: Biokinetic model; Hemoglobin A,,; Glucosylation

The reaction kinetics for the reversible condensation of D-glucose and haemoglo-
bin A through a labile haemoglobin A-aldimine adduct to HbA,, have been
investigated using a biokinetic model. The specific rate constants obtained from in
vitro experiments were included in the model which also took into account the
removal of HbA,, by decay of erythrocytes.
Using a sinusoidal variation in blood glucose a phase delay of about 2 hours was
observed between the maximum blood glucose concentration and the maximum
aldimine concentration. The mean haemoglobin A-aldimine concentration was inde-
pendent of both the amplitude and frequency of the blood glucose oscillations and
reached equilibrium concentration within 24 hours.
The steady state relation between mean blood glucose and HbA,, was similar to
the corresponding relation based on an irreversible formation of HbA,,. However,
contrary to the irreversible model the steady state HbA,, .concentration with the
reversible model was reached 3 to 4 weeks after a change in blood glucose level. This
finding is in agreement with clinical experience and indicates that in assessing
continuous glycaemic control in diabetic patients haemoglobin A,, should be
measured approximately every 3 to 4 weeks.

Glucosylation of the major human haemoglobin, haemoglobin A to haemoglobin

A,, (HbA,,) has been described as an irreversible process occurring slowly and

* Send reprint requests to: Hemik B. Mortensen, Department of Pediatrics, Glostrup Hospital, DK-2600
Glostrup, Denmark.

0009-8981/84/$03.C!O 8 1984 Elsevier Science Publishers B.V.

76

continuously throughout the 120-day life span of the red cell [l]. Determination of
HbA,, has been reported to provide a measure of the integrated blood glucose level
over the previous 2 to 3 months in diabetic patients [2,3]. However. in several recent
clinical studies it has been found that the concentration of glucosylated haemoglobin
only reflects the blood glucose level for a shorter period of time [4-61. In previous
studies [7,8] we have determined the rate and equilibrium constants governing the
formation of HbA,, through a labile intermediate, designated anodal gluco-
haemoglobin A, and it was shown that the formation of these glucosylated
haemoglobin species is reversible. These results have been included in a mathemati-
cal model which takes into account the removal of HbA,, by decay of erythrocytes.
Using this biokinetic model, the aim of the present study was to determine the rate
at which HbA,, approaches its equilibrium level at various physiological glucose
concentrations and further to investigate the length of the period during which a
single HbA,, determination reflects the mean blood glucose level.

Methods

The reversible condensation of D-glucose and haemoglobin A beta-chain terminal

valine is shown in the scheme. A Schiff base (haemoglobin A-aldimine) is formed
which in turn is capable of undergoing an Amadori rearrangement leading to the
formation of the ketoamine HbA,,.

k,=O.O96xlO-‘M-‘.s-’ k,=14,2~lO-~s-’
HbA + glc G=+ HbA-aldimine + HbA,,-ketoamine
k_,=o.lx10~‘s ’ k ~z=1.7X10-hS-’

The rate constants are given in the scheme [7,8]. The relative concentration of
HbA,, in a blood sample taken at a given time depends on the blood glucose level
over the previous period due to the relative slow attainment of equilibrium and the
continuous replacement of erythrocytes. A mathematical biokinetic model based on
the above reaction scheme and a constant life span of erythrocytes (120 days) is
described in the ‘Appendix’. This is an extension of a model developed by Beach [9]
based on irreversible HbA,, formation.

Results

In Fig. 1 the simulated relation between the relative concentration of the

HbA-alditnine and the mean blood glucose concentration is illustrated. A sinusoidal
variation in blood glucose concentration with a mean of 7.5 mmol/l, an amplitude
of 2.5 mmol/l and a frequency of l/3 to 3 day-’ was used. A phase delay of about
2 h is seen between the maximum aldimine concentration and the maximum blood
glucose concentration. The steady state mean HbA-aldimine concentration is con-
stant (0.7%) and independent of both the amplitude and the frequency of the blood
glucose oscillation. However, the amplitude decreased with increasing frequency of
blood glucose oscillations and the phase delay will approach l/4 of the full period.
 II

207 - Reversible
reortlons
,8_ --- lrrevers~ble
reorthons /
/
16. ,'
0
/
lL-

12-

lo-

a-

6-

L-

2-

I t 2 4 6 8 10 12 14 16 18 20
1 2 Constantglucoseconcentmtlon
d lmmol/ll

Fig. 1. The simulated variation of the HbA aldimine complex to sine wave variations in blood glucose
(mean 7.5, amplitude 2.5 mmol/l) are shown as the full line curves. The broken line curves show the
variation if instantaneous equilibrium were attained and further reflects the variation in blood glucose
concentration.

Fig. 2. The steady state concentration of HbA,, is calculated according to the reversible reaction system
using the rate constants mentioned in the text. For the simplified irreversible model rate constants of
Beach were used (upper curve k = 1.8 x 10m4 (l/mmo1)/24 h, lower curve k = 1.35 X low4 (l/mmol)/24 h.
An erythrocyte lifespan of 120 days was assumed in both cases.

Meon bloodglucose
ImmoVlj

HbAl
I% oftotalIHbl

0 15 30 45 60 75 90 105 lx) 135 150 165 lW 195 210 225 240

Days
Fig. 3. The simulated HbA,, variation in response to changes in blood glucose levels (upper curve). The
full line curve shows the HbA,, formation based on the reversible reaction scheme using the rate constant
given in the text. The broken line curve shows the HbA,, based on the irreversible reaction scheme
(k = 1.35 k 10m4 (l/mmol)/24 h). An,erythrocyte lifespan of 120 days was used for both simulations.
78

The reversible and the irreversible biokinetic models for the formation of HbA,,
were compared (Fig. 2). The steady state concentration of HbA,, was calculated
according to the reversible reaction system using the previously described rate
constants (scheme). For the simplified irreversible model rate constants of Beach [9]
were used (upper curve k = 1.8 X 10P4 l/mmol . 24 hh’. lower curve k = 1.35 x 10 4
I/mmol. 24 h-‘. An erythrocyte life span of 120 days was assumed in both cases.
The concentration of HbA,, as a function of time at different glucose concentra-
tions is simulated in Fig. 3. For the reversible model it is demonstrated that HbA,,
practically has reached the steady state within 3 to 4 weeks, independent of whether
a decrease or an increase in blood glucose level has occurred, whereas the irreversible
reaction system requires about 120 days to reach steady state. Consequently the two
models differ considerably when the blood glucose level changes within a 120-day
period.

Discussion

A mathematical model to predict glucosylated haemoglobin levels from retrospec-

tive blood glucose values was described by Beach [9]. This model was based on the
simplifying assumptions that erythrocytes have a life span of 120 days and that
glucosylated haemoglobin in vivo is irreversibly formed at a rate proportional to the
blood glucose concentration and the fraction of non-glucosylated haemoglobin. We
have developed an extension of this biokinetic model using the concept that the
Amadori rearrangement process is reversible.
From the simulated variation of the HbA-aldimine complex (Fig. 1) it is seen that
this compound reflects the mean blood glucose level within the last 24 h in the same
way as the HbA,, concentration reflects the mean blood glucose level for the
previous 3 to 4 weeks. Recently we have reported [lo] that a pronounced increase in
the HbA-aldimine occurs at HbA,, levels above 9 to 10% in insulin-dependent
diabetic children. Therefore the HbA-aldimine concentration is a useful index of the
integrated blood glucose concentration over the 24-h period prior to blood sampling.
Consequently this could be used as an alternative to blood glucose profiles obtained
by frequent blood glucose determinations.
It is seen (Fig. 2) that the steady state relation between mean blood glucose and
HbA,, for the reversible model was almost similar to the corresponding relation
based on the irreversible formation of HbA,,. Furthermore the predicted steady
state concentration for HbA,, in the reversible system at a given glucose concentra-
tion compares favourably with the experimentally determined HbA,, values in
insulin-dependent diabetic patients [ll].
For high glucose levels it is seen from the relationship between HbA,, and mean
blood glucose that a 1% change in HbA,, concentration corresponds to a change in
mean blood glucose concentration of 1.5 to 2.0 mmol/l. This finding emphasises the
need for an accurate and precise method for HbA,, determination [12] in order to
detect significant changes in the blood glucose profile.
It is seen that unlike the irreversible model the steady state HbA,, concentration
in the reversible model was reached 3-4 weeks after a change in blood glucose level
 19

(Fig. 3). This finding is in accord with the clinical observation that glucohaemoglo-
bin levels correlate well with blood glucose values for the preceding few weeks [4,5]
and that rapid changes in the ketoamine HbA,, can be observed during periods of
improvement or deterioration of glycaemic control [13,14]. It further indicates that
in assessing continuous glycaemic control in brittle diabetic children and adolescents
and in patients in whom intensified treatment regimes are to be monitored, HbA,,
should be measured approximately once every 3-4 weeks.
If the HbA,, concentration is determined less often, intermediate periods of
higher or lower blood glucose levels may not be detected. On the other hand, more
frequent measurement of HbA,, can only be expected to provide little additional
information of the glycaemic control because the HbA,, fraction can only respond
slowly to the more rapid variations in blood glucose level within this period.

Appendix

A biokinetic model for HbA,, as function of preceding blood glucose levels

Beach (1979) [9] has presented a kinetic model that describes the concentration of
glycosylated haemoglobin as a function of the previous blood glucose levels during
the life span of the erythrocytes. It is based on an irreversible formation of the
ketoamine (k_ z = 0) and additional simplifying assumptions, whereby the reaction
scheme was reduced to HbA + glucose + HbA,, with a single rate constant k = 1 x
1O-5 (mg/lOO ml)-’ . day-’ = 2 x 10P6 (mol/l)-’ . SC’. An extension of this model
based on the reversible reactions will briefly be given in the following.
Consider a cohort of erythrocytes of age t, and let a(t) and h(t) denote the
fractions of the total haemoglobin which are in the aldimine and ketoamine forms,
respectively. The following rate equations derived from the reaction scheme enable
a(t) and h(t) to be calculated from knowledge of their initial values and the glucose
concentration as function of time denoted g(t):

da(t)/dt=k,g(t)(l-a(t)-h(t))-k-la(t), (1)

dh(t)/dt=k,a(t)-k_,h(t). (2)

The general solution of these equations become rather complicated. Conse-

quently, simplified solutions that are approximately valid for the values of the rate
constants and glucose concentrations encountered in the application will be derived.
Since k_, is substantially larger than k, and k_ z the first reaction will be near
equilibrium provided g(t) does not vary too rapidly. This was studied by solving (1)
under the simplifying assumptions that a(t) +z 1, h(t) = 0, and for g(r) = g
+Asin(2rnt). The results obtained with a given mean blood glucose level (S), an
amplitude (A) and three different frequencies (n) are shown in Fig. 1. It is seen that
from an initial value a(0) = 0 a steady state is reached within 24 hours. For the
slowly varying g(t), n = l/3 day-‘, it is seen that near equilibrium is attained. For
the more rapid variation in g(t) it will appear from the following that h(t) is
determined by the mean level of a(t).
80

The equilibrium condition means that du(f)/dt = 0 and from (1) it follows that
a(t)=f(t)(l -h(t)) wheref(t)=k,g(t)/(k_, +k,g(t)). This is inserted into (2)
and the general solution for the equation for h(0) = 0 is:

where F(t) = j$( r )d=r. For contant g( t’) = g the equation becomes:

W = (W/q)(l - exp(-qf)L (4)

wheref= k,g/(k_, + k,g) and q= k_, + k2f.

Under the assumption that erythrocytes are formed at a constant rate and
eliminated after a constant life span (@), it follows that the mean relative HbA,,
concentration in a blood sample at time t0 is:

The argument of 6 means that 6 depends on all values of g(t) in the interval from
t, - 0 to to. For a constant g(t) = g it follows using (4) that

k(g)=(k,f/rl)fl-(l-exp(-qB))/qB). (6)
Fig. 2 shows hi(g) compared to the relation derived by Beach using the previously
stated rate constants.
The variation in h following a sudden change in g from one constant level to
another can also be calculated. If g(t) = go for t < 0 and g(t) = g, for t 2 0 it follows
that the values for z for t < 0 or t > 8 are given by inserting go or g,, respectively,
into (6). Hence it remains to find i for 0 < t < 8. If t’ denotes the age of an
erythrocyte cohort it follows from (3) in analogy with the derivation of (4) that:

(k,f,/q,)(1 -ex~(-q~f’))~O~t’<f
W’)= (k2fO/qO)[1--xp(-qo(~‘-~))]exp(-q,~) (9)
i +(k,f,/q,)(l - exp(--q,t)),tS f’s 8

and according to (5)

h(t,g,,g,)=(k,f,/q,)t(t/B)exp(-qlt)+(1-I/q,B)(1-exp(-q,t))l

+(k,.Wqo)exp(--q,t)[l -~/~-(l-exp(-rl,(~-t)))/q~~l,

o<ts,e. (10)
 81

Fig. 3 shows how HbA,, calculated according to (10) varies when g(r) is shifted
from one constant level to another. Although the true steady state level is not
reached until 0 = 120 days after a shift 6 is already very close to that level after 4
weeks. For comparison is also shown the variation in HbA,, calculated according to
the irreversible model derived by Beach. Although the steady state levels shown in
Fig. 2 are nearly identical, there are profound differences in HbA,, between the
reversible and irreversible reaction kinetics when the dynamic variation is consid-
ered. From Fig. 3 and equation (10) it also appears that kcannot respond to rapid
changes (frequency I 1 day-‘) in the glucose level. Hence, the mean blood glucose
level over periods of about 24 h or shorter will determine t? quite accurately.

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