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Artificial Organs

36(2):210223, Wiley Periodicals, Inc.


2011, Copyright the Authors
Artificial Organs 2011, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Thoughts and Progress


Impact of Hemoglobin Concentration and
Affinity for Oxygen on Tissue
Oxygenation: The Case of
Hemoglobin-Based Oxygen Carriers

[O2 ]a-v = [Hb] (Sa O2 S v O2 ) + (Pa O2 Pv O2 )


(1)
[Hb] and a represent the blood Hb concentration
and the O2 solubility coefficient (1.4 10-6 M/mm Hg
(2)), respectively. Although unlikely to accurately
yield the real D[O2]a-v, this relationship helps in
assessing how changes in a single variable reflect into
changes in D[O2]a-v. When the blood flow (Q) is
known or assumed, this relationship translates into
the Ficks equation, where VO2 represents the O2
consumption:

Michele Samaja and Laura Terraneo


Dipartimento di Medicina, Chirurgia e Odontoiatria,
Universit di MilanoSan Paolo, Milano, Italy
Abstract: In patients undergoing exchange-transfusion
with hemoglobin (Hb)-based oxygen (O2) carriers
(HBOC), native Hb coexists with newly transfused Hb.
The two Hb types share the same arterial and venous
PO2, but their affinities for O2 vary. A simple spreadsheet
model is described aiming at evaluating the contribution
of each Hb type to the overall O2 transport characteristics
as a function of the batch Hb concentration and O2 affinity in the HBOC solution, of the fraction of exchangetransfused blood/HBOC, and of the arterial PO2. This
model helps to yield a quantitative estimate of how
tissues with high or low O2 extraction respond to the
changes cited above. The results show that the higher the
exchange-transfusion ratio, the O2 transport to tissues
becomes progressively impaired. However, this effect is
more critical at low batch Hb concentration and high O2
affinity of the HBOC, especially for tissues/organs with
high O2 extraction, whereas the arterial PO2 does not
appear as critical. Key Words: HemoglobinOxygen
deliveryHypoxiaHemoglobin-based oxygen carriers.

VO2 = Q [O2 ]a-v

Whereas in a systemic situation Q and VO2 are,


respectively, the cardiac output and the body O2 consumption, with venous referring to the mixed
venous return, in a local situation within an organ or
tissue, Q and VO2 represent the blood flow and
organ/tissue O2 consumption, with venous referring
to the end-capillary compartment. In general, a moderate P50 increase tends to diminish SvO2 without
affecting considerably SaO2. This increases D[O2]a-v
favoring the O2 delivery when the other factors are
constant. For example, increasing blood P50 by
2 mm Hg allows either 10% Q reduction (less cardiac
load) or higher PvO2 (less tissue hypoxia) (3).
Hb-based O2 carriers (HBOC) are emerging as
potential substitutes for blood in emergency
situations. In the blood of patients transfused with
HBOC, Hb from HBOC (HbHBOC) coexists with
native RBC Hb (HbRBC). Despite sharing the same
PaO2, PvO2, and Q, the two Hbs differ in their relative
abundance ([HbHBOC] and [HbRBC]) and O2 affinity
(P50HBOC and P50RBC), hence SaO2 and SvO2. The twocompartment nature of such a situation can be
managed by the following equation:

Hemoglobin (Hb), a red blood cell (RBC) protein,


reversibly binds to the oxygen (O2) in the lungs to
release it to the tissues. The complex interaction with
CO2, 2,3-diphosphoglycerate (DPG), Cl- (for bovine
Hb), and protons determines the Hb affinity for O2
(1), which is conveniently expressed by its P50, or
PO2 at which half of Hb is bound to O2. By influencing the arterial and venous HbO2 saturations (SaO2
and SvO2, mole/mole) at any arterial and venous O2
partial pressure (PaO2 and PvO2), the HbO2 affinity
contributes to tune the arteriovenous O2 difference
(D[O2]a-v):

[O2 ]a-v = [HbRBC ] (Sa O2RBC S v O2RBC ) + [HbHBOC ]


(Sa O2HBOC S v O2HBOC ) + (Pa O2 Pv O2 )
(3)

doi:10.1111/j.1525-1594.2011.01296.x

With some considerable exception, the various


HBOCs frequently display a high HbO2 affinity,
with P50 as low as 5 mm Hg. This feature is perceived
negatively because the high HbO2 affinity is predicted to impair the O2 delivery: HbHBOC binds to the

Received July 2010; revised February 2011.


Address correspondence and reprint requests to Professor
Michele Samaja, Dipartimento di Medicina, Chirurgia e Odontoiatria, San Paolo, Universit di Milano, Via di Rudin 8, Milano
20142, Italy. E-mail: michele.samaja@unimi.it

210

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(2)

THOUGHTS AND PROGRESS


O2 strongly in the lungs and releases it slowly to the
tissues. In the lack of a suitable HBOC to address this
issue experimentally without overlapping phenomena such as vasoconstriction, extravasation, increased
systemic vascular resistance, and methemoglobin formation, the computer simulation object of this report
uses the two-compartment modeling of Eq. (3) to
predict the effects of changing some selected variables on D[O2]a-v.

METHODS
To implement the model, we used the O2 equilibrium curves (OECs) of HbRBC and propositus HbHBOC
with a PO2 resolution of 0.25 mm Hg in the range
0100 mm Hg. The OEC for HbRBC was obtained
assuming normal values for CO2, pH, and DPG
(P50 = 28 mm Hg) (4).The OECs for a high and a low
O2 affinity HbHBOC (MP4 and aaHb, P50 = 5 and
30 mm Hg, respectively) were provided from
Sangart, Inc. (San Diego, CA, USA). The OECs for
HBOCs with P50 = 20 and 40 mm Hg were obtained
from intrapolation/extrapolation of the mentioned
ones.
D[O2]a-v was calculated from Eq. (3) via an Excel
spreadsheet freely available from the authors. The
inputs are the OECs for HbRBC and HbHBOC and
their blood Hb concentrations [HbRBC] and
[HbHBOC]. The total O2 content (chemically bound to
Hb plus physically dissolved) in the arterial blood
([O2]a) at the selected PaO2 is calculated from the
OEC as:

[O2 ]a = [HbRBC ] Sa O2RBC + [HbHBOC ] Sa O2HBOC +


(4)
Pa O2
The calculation of the total O2 content in venous
blood ([O2]v) is subject to experimental limitations
for the known obstacles in measuring end-capillary
and mixed venous return PvO2. Therefore, a
matrix of [O2]v values is generated as a function of
PvO2 in the range 0-PaO2 by the following equation,
where SvO2 is obtained at each PvO2 using the OEC:

[O2 ]v = [HbRBC ] S v O2RBC + [HbHBOC ] S v O2HBOC +


Pv O2
(5)
Finally, D[O2]a-v is calculated at any PvO2 and [O2]v:

[O2 ]a-v = [O2 ]a [O2 ]v

(6)

Therefore, the final matrix reports D[O2]a-v as a


function of PvO2 in the range 0-PaO2 mm Hg.

211
RESULTS

For model testing, consider the case of a 70-kg


patient
undergoing
isovolumetric
exchangetransfusion (XT) with MP4 (P50 = 5 mm Hg) with
batch [Hb] = 4 g/dL.The XT ratio varies from 0% (no
XT) to 100% (blood entirely replaced by HBOC).
[HbHBOC] and [HbRBC] are calculated at each XT ratio
assuming blood volume = 5 L. Figure 1 reports the
OECs (panel A), the total blood O2 content (panel
B), and the relationship between D[O2]a-v and PvO2 at
varying XT ratios (panel C). An interpretation
follows. When PvO2 increases, SvO2 approaches SaO2,
and hence, D[O2]a-v tends to zero. By contrast, when
PvO2 approaches 0, D[O2]a-v depends on total [Hb], or
[HbHBOC] + [HbRBC], because O2 is unloaded independently of the HbO2 affinity. At constant batch
[HbHBOC] and blood volume, increasing XT ratio
decreases [HbHBOC] + [HbRBC], and hence D[O2]a-v. At
intermediate PvO2, D[O2]a-v depends on [Hb] and P50.
Should all Hb occur as HbHBOC (XT = 100%) and
P50HBOC is low, then a very low PvO2 is needed to
unload O2. When Hb is present as both HbRBC and
HbHBOC, an intermediate situation occurs. As a whole,
when batch [Hb] and P50HBOC are low, a progressively
lower PvO2 is needed to extract O2 with increasing
XT ratio.
Figure 2 predicts D[O2]a-v, assuming the above case
and after varying systematically one of the following
variables: batch [Hb] in HBOC, which reflects into
varying [HbHBOC] (panel A), P50HBOC (panel B) and
PaO2 (panel C), while keeping constant the XT ratio
(50%, hence [HbRBC]) and P50RBC. As all the variables
affect D[O2]a-v, the knowledge of PvO2 represents the
most crucial point in this modeling.
As it is intuitive that PvO2 depends primarily on
tissue O2 extraction, a likely value for PvO2 was
derived from the reported tissue/organ VO2, Q, and
D[O2]a-v (5), assuming a resting male subject with
VO2 = 0.25 L/min, PaO2 = 95 mm Hg, P50RBC = 28
mm Hg, and blood [Hb] = 16 g/dL. The estimated
PvO2 was then used to interpolate D[O2]a-v in Fig. 2.
Figure 3 reports the assumed PvO2 values and the
impact of [HbHBOC] PaO2 and P50HBOC in the considered organs while maintaining constant the XT ratio
(50%), [Hb]RBC, and P50RBC. It appears that organs
with high O2 extraction respond to the presence of
HbHBOC in the blood by keeping lower PvO2 organs
with low O2 extraction. Thus, in patients undergoing
XT with HBOC, high O2 extraction organs suffer a
higher degree of hypoxia than low O2 extraction
organs. Furthermore, if the slope of the curves
indexes the interdependence of the variables
represented on the axes, it appears that the
Artif Organs, Vol. 36, No. 2, 2012

212

THOUGHTS AND PROGRESS

O2 equilibrium curve

HBOC

0.6
0.4

Blood

50
PO2, mm Hg

75

6
4

100

10

50
5.0

75

100

40
28
20
10
5

P50RBC=28 mm Hg
[Hb]HBOC=2 g/dl
[Hb]RBC=8 g/dl
PaO2=95 mm Hg

6
4

75

P50HBOC, mm Hg

100

2.5

25

100

50

75

100

PvO2, mm Hg
C

O2 delivery

10

0
25
50
75
100

XT=0-100%
P50HBOC=5 mm Hg
P50RBC=28 mm Hg
[Hb]HBOC=0-8 g/dl
[Hb]RBC=16-0 g/dl
PaO2=95 mm Hg

8
6
4

PaO2, mm Hg

10
XT=50%
P50HBOC=5 mm Hg
P50RBC=28 mm Hg
[Hb]HBOC=2 g/dl
[Hb]RBC=8 g/dl
PaO2=55-95 mm Hg

8
6
4

95
85
75
65
55

2
0

O2 delivery

XT, %

[O2]a-v, mM

[O2]a-v, mM

50
75
PvO2, mm Hg

XT=50%
P50HBOC=5-40 mm Hg

8
[O2]a-v, mM

25

7.5

50
PO2, mm Hg

25

O2 delivery

10.0

25

XT, %

Total [O2], mM

0
25

O2 equilibrium curve

0.0
0

8
6
4
2
0

XT=50%
P50HBOC=5 mm Hg
P50RBC=28 mm Hg
[Hb]HBOC=0-6 g/dl
[Hb]RBC=8 g/dl
PaO2=95 mm Hg

0.2
0.0
0

[HbHBOC], g/dl

10
[O2]a-v, mM

Hb-O2 saturation

0.8

O2 delivery

P50, mm Hg
5
28

1.0

25

50
PvO2, mm Hg

75

100

FIG. 1. O2 delivery in a 70-kg patient with blood volume = 5 L,


undergoing exchange-transfusion (XT) with high O2-affinity
HBOC. (A) O2 equilibrium curves (SO2 vs. PO2) of HbRBC
(P50 = 28 mm Hg) and HbHBOC (P50 = 5 mm Hg). (B) Total O2
content vs. PO2 assuming isovolumetric XT (batch [Hb] = 4 g/dL)
for 0% (no XT), 25%, 50%, 75%, and 100% (all blood replaced
with HBOC). [HbRBC] and [HbHBOC] are thus varied stepwise
16-to-0 and 0-to-8 g/dL, respectively. (C) O2 delivery, or [O2]a[O2]v, assuming PaO2 = 95 mm Hg.

25

50

75

100

PvO2, mm Hg
FIG. 2. O2 delivery at varying batch [Hb] and P50 in the HBOC
and oxygenation state. (A) Effect of varying [HbHBOC] in the range
08 g/dL in the circulation, which is equivalent to 012 g/dL in
the batch, at constant P50RBC, P50HBOC, [HbRBC], and PaO2 (see
inset). (B) Effect of varying P50HBOC in the range 540 mm Hg at
constant P50RBC, [HbHBOC], [HbRBC], and PaO2 (see inset). (C)
Effect of varying PaO2 in the range 5595 mm Hg at constant
P50RBC, P50HBOC, [HbHBOC], and [HbRBC] (see inset).

DISCUSSION
positive effect of increasing [Hb]HBOC is evident only
when P50HBOC is high. By contrast, PaO2 does not
appear as crucial because its value lies above the
PO2 range where O2 unloading by Hb might have
any influence.
Artif Organs, Vol. 36, No. 2, 2012

The model is limited by the following assumptions:


1 Absence of changes in the ventilation rate, or constant PaO2. However, the effect of varying PaO2 on
D[O2]a-v does not appear as relevant as those due to
changes in other variables.

THOUGHTS AND PROGRESS

213

A
P50HBOC=5 mm Hg

P50HBOC=40 mm Hg

80

Kidneys

PvO2, mm Hg

70
60
Total
Brain
Legs
Heart

50
40
30
20
10

8 0

[HbHBOC], g/dL

[HbHBOC], g/dL

B
P50HBOC=5 mm Hg

P50HBOC=40 mm Hg

80
Kidneys

70

PvO2, mm Hg

60
50

Total
Brain
Legs

40
30

Heart

20
10

50

60

70

80

90

50

60

70

80

PaO2, mm Hg

PaO2, mm Hg

[Hb]HBOC=1 g/dL

[Hb]HBOC=4 g/dL

90

FIG. 3. O2 delivery in various organs. (A)


Effect of [HbHBOC] on the PvO2 needed to
deliver the amount of O2 indicated in the
text at constant values of [HbHBOC] (4 g/
dL), [HbRBC] (8 g/dL), P50RBC (28 mm Hg),
and PaO2 (95 mm Hg). P50HBOC is either 5
(left) or 40 mm Hg (right). (B) Effect of
PaO2 on the PvO2 needed to deliver the
amount of O2 indicated in the text at constant values of [HbHBOC] (4 g/dL), [HbRBC]
(8 g/dL) and P50RBC (28 mm Hg). P50HBOC
is either 5 (left) or 40 mm Hg (right). (C)
Effect of P50HBOC on the PvO2 needed
to deliver the amount of O2 indicated
in the text at constant values of PaO2
(95 mm Hg), P50RBC (28 mm Hg), and
[HbRBC] (8 g/dL). [HbHBOC] is either 1 (left)
or 5 g/dL (right). The assumed values
for PvO2 for brain, heart, kidneys, legs,
and total body are 44, 34, 70, 41, and
47 mm Hg, respectively.

80

Kidneys

70

PvO2, mm Hg

60

40

Total
Brain
Legs

30

Heart

50

20
10

10

20

30

P50HBOC, mm Hg

40

10

20

30

40

P50HBOC, mm Hg

2 Constant level of the Hb allosteric factors pH,


PCO2, 2,3-DPG, and Cl- (for bovine Hb) throughout the observation, which translates into constant
P50RBC and P50HBOC. In human blood under stan-

dard conditions, P50 is relatively constant but subjected to fluctuations arising in response to changes
in the mentioned allosteric effectors as well as
other factors that determine the actual P50 in vivo,
Artif Organs, Vol. 36, No. 2, 2012

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THOUGHTS AND PROGRESS

including temperature, hydrostatic pressure, intracapillary Bohr effect, and varying RBC distribution (e.g., old and young RBCs coexisting in
the same blood stream) (6). Some of these changes
might, in principle, affect P50HBOC as well.
No loss of HBOC or RBC from the circulation,
or constant [HbHBOC] and [HbRBC], whereas Hb
extravasation is known to be a critical phenomenon in HBOC experimentation.
Negligible effects of nitric oxide (NO), an
endothelium-derived relaxing factor with a
number of effects in somatic cells. NO stimulates
soluble guanylate cyclase to form cyclic guanosine
monophosphate (cGMP), a messenger that mediates a large number of effects in the microcirculation in vivo. Current investigations also address the
hypothesis that Hb is a NO transporter (7) that
responds to changes in the Hb oxygenation state
(8) and helps NO export out of the RBC promoting local vasorelaxation and improving VO2 upon
Hb deoxygenation (9).
Constant RBC capillary transit time with negligible
blood flow autoregulation. However, recent work
addressed cerebral blood flow autoregulation as a
sensitive mechanism to tune the blood O2 transport
in response to the body/organ needs (10,11).
The described model cannot assess where exactly
O2 unloading in the capillary bed occurs, e.g., in the
precapillary area or elsewhere, while it is well
appreciated that O2 unloaded in the precapillary
contributes to vasoconstriction rather than tissue
oxygenation (12).

It is worthwhile to note that the unavailability of in


vivo data obtained with HBOC in the place of human
blood led to plot Figs. 1C and 2 in a PvO2 range that
exceeds the range of clinically or physiologically relevant values.
Inadequate O2 delivery invariably leads to hypoxia,
which might be lethal if severe and/or sustained.But in
the case of sublethal hypoxia, cells are enabled to
resist by overexpression of the hypoxia-inducible
factor 1a (HIF-1a), a ubiquitarian molecule that regulates the transcription of a variety of downstream
genes coding for proteins that elicit hypoxia adaptation (13). Besides being a hypoxia marker, HIF-1a is
thus pivotal to orchestrate the cell response to
hypoxia.Although this feature is not shared equally by
the body organs (14), HIF-1a accumulates in hypoxic
tissues in vivo, and such accumulation might be critical
to elicit a useful response to hypoxia, as, for example,
triggering angiogenesis, resisting to apoptosis, increasing the antioxidant defense, and others. It is thus a
matter for further investigation whether tissue
Artif Organs, Vol. 36, No. 2, 2012

hypoxia must be avoided or may instead represent a


way to induce resistance to stress. Most likely, lack of
O2 is not the only trigger for HIF-1a overexpression,
and not all the responses triggered by hypoxia are
mediated by HIF-1a. Nevertheless, the HIF-1a
pathway is prominent in determining the cell/tissue
ability to respond to hypoxia, and its overexpression
may be crucial to allow greater protection in HBOCperfused tissues. It was observed that rats XT with a
polymerized bovine Hb with P50 of 46 mm Hg and
stock Hb concentration of 13 g/dL display attenuated
HIF-1a response in the kidney (15), despite increased
HIF-1a in hypoxic bovine aortic endothelial cells
incubated in the presence of diaspirin cross-linked Hb
(16).That outcome was predicted by the present modeling (right plot in Fig. 3C), as kidneys perfused under
comparable conditions are far from being hypoxic,
thereby providing an explanation for the low HIF-1a
response in that organ.
CONCLUSION
The mathematical model enables predicting how
batch [Hb] and P50 in HBOC, as well as the PaO2,
cooperate in tuning the O2 delivery to tissues in
patients undergoing XT with HBOC.The results show
that the paradigm the higher the HBOC-O2 affinity,
the less efficient O2 delivery becomes might sometimes be an oversimplification. If the slopes of the
curves in Fig. 3 provide an indication of the dependency of the O2 delivery on either [Hb]HBOC or PaO2, it
appears that batch [Hb] becomes a critical factor only
when P50HBOC lies in the upper range. The PaO2
appears less critical than batch [Hb] and P50. Furthermore, the O2 extraction by the single organ represents
an additional factor to be considered because organs
with high O2 extraction as the heart and brain are
predicted to respond to [Hb]HBOC and P50HBOC to a
minor degree than organs with low O2 extraction as
the kidneys because of fluctuations in the local PvO2.
In conclusion, it is not only the P50 that matters, but
rather, the complex interaction among P50, [Hb], and
PvO2 in the various organs that need to be taken into
account when assessing the O2 delivery efficiency of
HBOC.
Acknowledgments: We thank Drs. Kim Vandegriff
and Ashok Malavalli (Sangart, Inc.) for helpful discussion and providing O2 affinity data related to MP4
and aaHb.
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