35
Clinical Science (I997)94,35-41 (Printed in Great Britain)
Plasma oxygen during cardiopulmonary bypass: a comparison of
blood oxygen levels with oxygen present in plasma lipid
1. M. PETYAW*, A. WYLSTEKEt, D. W. BETHUNEt and J. V. HUNT*
*Division of Chemical Pathology, Clinical Sciences Building, The Glenfield Hospital - NHS Trust, Groby
Road. Leicester lE3 9QP. U.K.. and tPapworth Hospital NHS Trust, Department ofhaesthesia, fapworth
Everard, Cambridge CB3 8R€, U.K.
(Received 26 JuneR2 August 1997; accepted I September 1997)
1. Although not often appreciated, it is a fact that
molecular oxygen is more soluble in lipids than in
aqueous solution, We have recently developed a
method to monitor oxygen within the lipid content
of plasma. Monitoring plasma oxygen is one essential element during open heart surgery using a
cardiopulmonary bypass pump and oxygenator.
Currently oxygen is monitored electrochemically
and is based upon monitoring the partial pressure
of oxygen in a gas equilibrated with whole blood.
2. To determine the relative importance of lipidassociated oxygen in blood and assess the potential
use of such a measurement we present comparisons
of changes in oxygen associated with whole blood
and lipid content of plasma before, during and after
cardiac surgery.
3. In a limited number of patients studied (n = 28),
aged between 34 and 86years, oxygen in lipid
increased with decreased extracorporeal blood temperature during cardiopulmonary bypass, increased
in proportion to oxygen supplied and appeared to
be a better monitor of oxygen than conventional
electrochemical systems currently in use. Oxygen
associated with whole blood and plasma lipid was
markedly below normal on aortic declamping after
cardiopulmonary bypass, suggesting an hypoxic
episode at this point. Levels of oxygen in the lipid
phase of plasma returned to normal presurgical
values 6-8 h after surgery.
4. Calculation of the concentration of lipid-associated oxygen present in plasma suggests that plasma
lipids contain up to 25% of that typically ascribed to
haemoglobin. Thus, we suggest that monitoring
lipid-associated oxygen may prove a better alternative to current methods of measuring oxygen status.
Furthermore, we suggest that plasma lipid is a
hitherto unsuspected pool of circulating oxygen
Key words: Cardiopulmonary bypap, l i d , oxygen.
A b b d a t h CPB. Cardiopulmonary bypap.
corray#ndaner.Dr I. V. Hunt
which may play a significant role in tissue oxygen
supply.
INTRODUCTION
The postperfusion syndrome plays an important
role in the morbidity and mortality of patients who
have undergone cardiac surgery. Cardiac dysfunction
after cardiopulmonary bypass (CPB) has been
reported by various investigators and abnormalities
of post-operative ventricular function can occur
even after uneventful operations [l-61. Such surgery
is accompanied by significant disturbances of oxygen
and its metabolism both in the myocardium and in
the blood passing through CPB 17-91. Indeed, it is
known that CPB with a pump oxygenator is associated with free-radical-mediated damage [lo-121
which may result from leucocyte activation [9],
arachidonic acid metabolism [131 and/or lipid peroxidation [lo-12, 141. This damage appears to continue well after surgery [9, 15, 161. Thus, monitoring
the level of oxygen present in the circulation is
important during cardiac surgery using a CPB pump.
Hypoxia is known to be detrimental but controversy
exists with respect to the upper acceptable limits of
blood oxygen. To date, no indication of upper limits
exists in adult patients during extracorporeal oxygenation.
Traditionally, the oxygen content of blood is measured by means of a Van Slyke apparatus 1171.
Using this method, the blood is haemolysed and the
contained gas extracted by vacuum. Oxygen is then
chemically absorbed and the oxygen content calculated from the resulting fall in gas pressure. Currently, clinical assessment of blood oxygen levels is
based upon electrochemical detection of the partial
pressure of oxygen in a gas equilibrated with blood
[18]. Haemoglobin saturation can either be inferred
36
I. M. Petyaev et al.
from the partial pressure or measured by subsequent spectrophotometric means.
It is known that oxygen has an increased affinity
for hydrophobic phases. The concentration of oxygen in lipid may be an order of magnitude greater
than in the aqueous phase [19-221. Thus, a major
pool of oxygen in blood may be lipidsflipoproteins
which, to date, has largely defied measurement in a
clinical context. We have recently developed a
simple colorimetric method of monitoring oxygen
quantitatively in aqueous mixtures containing lipid
micelles, lipid emulsions and artificial lipoproteins.
The same method can also monitor the oxygen content of lipidflipoproteins in whole plasma and purified low-density lipoprotein. This method, described
in detail elsewhere, is based upon linking a monitored oxygen-dependent reaction with oxygen in
lipid components [19].
This study involved the prospective examination
of 28 patients undergoing CPB and extracorporeal
oxygenation during cardiac surgery. For the first
time, we present a comparison of changes in blood
oxygen with oxygen present within the lipid content
of blood plasma, monitored by a recently developed
technique based upon the contribution of lipidderived oxygen to micellar catalysis of an oxygendependent reaction.
MATERIALS AND METHODS
Cardiopulmonary bypass
The patient group, containing both smokers and
non-smokers lacking any obvious pulmonary disease,
consisted of 6 women and 22 men aged between 34
and 86 years. Patients selected displayed no obvious
abnormality in lipoprotein profile although cholesterol levels were slightly elevated; 6.3 k0.5 mmol/l.
The majority underwent coronary artery bypass graft
surgery (23 patients) and the remainder valve
replacement surgery (5 patients). All patients gave
informed consent for drawing blood and the trial
was approved by the Local Research Ethics Committee.
A standard anaesthetic regimen was used, mainly
based on a combination of an infusion of methohexital (3 mg h-'kg-I), a loading dose of fentanyl
(0.015mgkg) and a bolus of pancuronium (0.1
mgkg). Atracurium (0.75 mgkg) was added at the
start of bypass. Isoflurane (0.5-1%) or sodium nitroprusside were used to control blood pressure. Morphine was given post-operatively. The CPB circuit
was primed with a 1.15 litre Hartmann solution and
350 ml of mannitol (10%). A membrane oxygenator
was used and extracorporeal blood was cooled to
29-31°C.
At various times during the surgical procedure
blood was drawn through a catheter fixed in the left
radial artery. Inspired oxygen concentration was
continuously monitored using a Datex capnograph
during the pre- and post-bypass phases of surgery.
Ventilation settings were adjusted as a function of
the end-tidal C02 and airway pressures. Oxygen flow
was adjusted and the gas supplied was a mixture of
oxygen and air.
Sampling procedure
During blood sampling, six 0.1 ml samples were
added to 0.9 ml of 50 mmolfl potassium phosphate
(pH 7.8) containing 1 mmolfl diethylenetriamine
penta-acetic acid. Samples were mixed and centrifuged, and plasma was stored at room temperature
before analysis within 2-3 h.
Oxygen measurement
Conventional electrochemical measurement of the
partial pressure of whole-blood oxygen was determined immediately after sampling by means of a
polarographic oxygen electrode.
The level of oxygen in lipidflipoprotein present in
plasma was determined as described elsewhere [23].
The assay is a simple chemical one and is based
upon monitoring an oxygen-dependent reaction
spectrophotometrically over a period of minutes.
Although several similar chemical systems have been
investigated, in these studies the measurement of
nitrobluetetrazolium-detectable superoxide generated by NADH and phenazine methosulphate in the
presence of diethylenetriamine penta-acetic acid was
used. The assays were performed in 96-well plates.
Previous studies have shown that oxygen in lipid/
lipoprotein present in plasma is relatively stable with
a loss of <3% over 3 h and a loss of < 10% over
2 days (results not shown).
Plasma cholesterol and protein content were
monitored spectrophotometrically as described previously [24, 251. Studies of the oxygen content or
human low-density lipoprotein in vitro were also
conducted, as described previously [23].
Data analysis
Graphics, with lines of regression (linear or polynomial), were drawn on Cricket Graph. All statistical analyses were performed using Microsoft Excel
4.0 (Apple Macintosh).
RESULTS
Lipid-associatedoxygen before CPB
Oxygen in plasma lipid increased linearly with
total plasma cholesterol levels, as shown in Fig. 1.
Plasma oxygen, lipid and bypass
Furthermore, plasma lipid-associated oxygen
appears to be linearly related to the percentage of
oxygen supplied (Fig. 2, top). On the other hand,
electrochemical measurements of oxygen were linear
only over a limited range of oxygen supplied (Fig. 2,
bottom).
31
(Table 1). After induction of narcosis, the inspired
level of oxygen was reduced from 90-100% to 60%.
The level of lipid-associated oxygen also decreased
(Table 1). Electrochemical measurements of oxygen
failed to reflect this relatively rapid drop in oxygen
supply (Table 1). During CPB, the perfusionist maintained pre-operative oxygen levels by altering the
flow ratio of oxygen to air, as assessed electrochem-
Lipid-associated oxygen during extracorporeal blood
supply
Before narcosis an initial inhalation of 40% oxygen gas was used. This was slowly increased to
90-100% just before narcosis. During this period
there is an increase in lipid-associated oxygen and
also in the electrochemical detection of oxygen
"
I
0
-
I
20
.
I
40
.
I
60
.
,
.
80
I
.
1 0
100
% Oxygen in inhalation mixture
201
0
-6.
r = 0.95 (polynomial)
,
20
40
. r =I 0.88
. (linear)
, .
60
80
I
.
100
120
% Oxygen In inhalation mixture
4
5
6
7
8
Fe. 2. Canporkon of decbocherml
' dacaionmdmuwrsmentof
[Cholesterol]
Fig. I . h
(mM/L)
a chdestsrd and plasma lipkl-luodrtld oxygen. The
relationthip between plavM lipidderived oxygen and total cholesterol.Each
value of lipidderived oxygen was obtained from 3-5 patients with a cholesterd level within 7%. Values are means+SD. Assays were performed in
triplicate.
lipid-ruocirtcd oxygen. The meawrement of plma lipid-associated oxygen (top) and partial prersure of oxygen (bottom) as a function of administered oxygen. FW each due. data are from 6-15 p
a
w
.Valuet are
meansfSD. Data has been extapdated through the origin to demonstrate
the linear relationship between meawtwnents of lipid-arrodated oxygen and
inhaled oxygen. Eleamchemicd mea~ementfwere not linearly related to
administered oxygen. k a y s were performedin t r i p l i i .
I. M. Petyaw et al.
38
Table I. Blood oxygen in patienk undegOing open heart s u ~ e r y .Units of oxygen in lipid/lipoprotein present in plasma are expressed in lo-' moln oxygen per mg
of plasma protein. Blood oxygen was also monitored by conventional electmhemical m e ~ (unio;
s
kPa). Siiifkant changes from preopeative values were tested by
analym of m i m e .
stage of operation
Pre-operatiw
Induction of narcosis
Prebypass
CPB
End of CPB
End of surgery
6-8 h after
20-22 h later
% oxygen gas supplied
40
90-100
60
w
85-95
55-60
60
hbem (2096)
2.18 k0.135
28
4.6k0.4
23
<0.001
2.0k0.3
IS
<0.05
4.64k0.35
22
<0.001
0.97kO.l
21
<0.00I
1.57k0.05
20
<0.00I
2.0 f 0. I 7
12
>0.05
2.07 k0. I3
7
>0.05
49.7 3.0
I2
<0.005
38+ 1.0
10
>0.05
36.6k0.77
25
>0.05
27.4k1.85
8
29.1 k 1.3
I1
k0.03
7
>0.05
Lipidassociated oxygen
(IO-~~O
I-'I mg-' protein)
M a n k5D
n
P value
Oxygen in whde blood
(kP3
Mean f 5D
n
36.8
10
+2.9
P value
I2k2.0
<0.00 I
II
<0.02
35k1.0
*Thr~@ brpars oxygenator. w
nadjusted to prpoperative partial pressure, by altering oxygenlair ratio and flow.
ically. Despite this, measurements of lipid-associated
oxygen suggest that the blood is fully oxygenated,
resembling levels present on supplying a patient with
90-100% oxygen gas during the initial phase of narcosis (Table 1). Thus, a discrepancy exists between
these two measurements.
Cooling of extracorporeal blood during CPB had
a significant influence on the content of lipid-associated oxygen (Fig. 3A). Oxygen content appears to
be greater at low temperature (28-30°C). Above
31°C lipid-associated oxygen, obtained from patients
before CPB, decreased significantly. Studies of puri-
B
A
rr0.95
90
-
80
-
I
A
'O-
m
E
\
60-
z
;,I
Y
r
2
.
40-
2
.
30
20
26
20
30
32
34
Temperature (oC)
36
30
f-
28
II
36.7
Temperature (oC)
Fig. 3. TempMture effects on plasma lipid-wodated oxygen during CPB. The effect of tempetature upon (A) plasma lipidassociated oxygen and (B) lipid-associatedoxygen of purified human lowdensity lipoprotein (LDL). To determine the effect of temperature, levels of oxygen were analysed in patients befcfe CPB and during CPB. Each value was obtained from 3-5 patients. Values
are means+SD. Assays were performed in triplicate. Oil. oxygen in plasma lipid.
39
Plasma oxygen, lipid and bypass
fied human low-density lipoprotein in v i m showed a
similar effect of temperature (Fig. 3B).
Lipid-associated oxygen after CPB
In the first 3-9 min after CPB plasma lipid-associated oxygen and electrochemical measurements of
oxygen dropped to less than 60% of plasma levels
before surgery (Table l), despite a supply of 100%
oxygen gas. Figure 4 shows the CPB levels of plasma
lipid-associated oxygen as a function of bypass duration both immediately after CPB (A) and 6-8 h
later (B). Table 1 shows that there is a return to
presurgical levels of blood lipid oxygen content after
surgery within 6-8 h after surgery.
DISCUSSION
Oxygen in plasma lipid appears to be linearly
related to total plasma cholesterol (Fig. 1) and
increases in oxygen supply lead to an increase in
lipid-associated oxygen (Fig. 2, top). The true significance of oxygen within the lipid components
present in blood has yet to be examined. In our
opinion, it would not be at all surprising that lipids
constitute a major route of oxygen supply to tissue.
After all, even haemoglobin releases oxygen into the
aqueous phase of blood. Once in the blood the oxy-
\
gen must gain access to tissues and eventually to the
intracellular environment; it can do this relatively
slowly by diffusion alone. Alternatively, oxygen can
enter blood lipidsflipoproteins where it is far more
soluble. Blood lipidflipoprotein components might
serve as ‘packages of oxygen’ which may interact
with membranes better than water itself. The
aqueous phase of plasma may even be considered an
insulator as exemplified by the stability of oxygen in
the lipid fraction of plasma (results not shown). A
question of access might also apply. Blood plasma
and its contents may gain access to places and
spaces inaccessible to the haemoglobin-containing
erythrocytes. These observations raise an interesting
question of whether or not the control of plasma
lipid content, in common practice for patients with
cardiovascular disease, is wise in prospective CPB
patients. Low lipid levels may reduce tissue oxygen
supply whereas high levels might accelerate free
radical production. Given the detrimental effects of
increased plasma lipid in cardiovascular disease and
the reported benefits of plasma lipid reduction, a
more detailed investigation of plasma lipid content,
changes in lipid-associated oxygen during surgery
and the recovery rate of the patients after surgery is
essential before any valid assessment can be made.
Assuming equilibration between 20% oxygen gas
and an aqueous system, water-soluble oxygen
amounts to 2.4 x
molfl. Oxygen dissolved in
r=0.93
-
20
40
60
60
Duration of CPB (Minutes)
I
1
40
.
1
60
-
1
80
-
1 0
Duration of CPB (Minutes)
Fie. 4. CPB duration d phsma lipkksodated oxygen. Plasma oxygen in lipidllipoprotein present in plasma at (A) the end of
the opentkn and (B) 6-8 h after. Plasma lipid-associated oxygen appears to decraace with duration of CPB but seems to m m to
near-normalii with time after s
um.Each value was obtained from 3-5 patients. Values are meanrfSD. h a y s were performed in
triplicate. Oil. oxygen in plasma lipd.
40
I. M. Petyaev et al.
blood water is assumed to account for approximately
1.5% of total blood oxygen; the rest is assumed to
be in haemoglobin. On the assumption that the
aqueous content of blood reflects that of water equilibrated with ambient oxygen tension, haemoglobin
contains 0.016 molfl oxygen. Our measurements of
lipid-associated oxygen, when patients inhale 20%
oxygen, amount to 1.2 x
molfl oxygen per mg
of plasma (see Table 1 or Fig. 2) which is equal to
0.0042 molfl oxygen per litre of plasma (plasma typically contains 25 mg/ml protein). Thus, up to 25% of
total oxygen present in whole blood is within the
lipid fraction of plasma.
A comparison of the measurement of lipid-associated oxygen with the conventional electrochemical
system for measuring oxygen levels suggests that the
measurement of lipid-associated oxygen is a better
reflection of the oxygen supplied. In other words,
lipid-associated oxygen is linearly related to inhaled
oxygen supply (Fig. 2, top), whereas this linearity
only occurs over a very limited range in the case of
electrochemical detection (Fig. 2, bottom) [ 181. This
lack of linearity for the electrochemical assessment
of blood oxygen may be due to longer times of equilibration and mechanical and/or electrical limitations of electrochemical detectors [ 181.
In the pre-bypass period, inhaled oxygen was
reduced from 90-100% to 50% inhaled oxygen
(Table 1 and Fig. 3). Measurements of lipid-associated oxygen reflected this reduction better than the
electrochemical measurements. Again there would
appear to be a differing sensitivity of the two
measurements. It is possible, but unlikely, that the
period before CPB is relatively short (up to 45 min)
and the discrepancy between levels of lipid-associated oxygen and electrochemical detection may
occur as a result of differences in diffusion rates of
oxygen in aqueous (electrochemical detection) and
hydrophobic lipid environments (lipid-associated
oxygen detection). In other words, diffusion of oxygen into lipids is faster and thus levels of lipid-associated oxygen respond more rapidly to changes in
inhaled oxygen supply.
Although the solubility of oxygen in aqueous
systems, and in organic solutions too, is decreased
with a drop in temperature, the effects of temperature shown in Fig. 3 may be due to changes in lipid
fluidity. Noteworthy is the 31°C phase transition
temperature of lipids which also appears to be the
transition between differences in plasma lipid-associated oxygen with temperature (Fig. 3A). Studies of
lipid-associated oxygen in purified low-density lipoprotein in vitro suggest that the temperature effect is
indeed due to changes in lipid fluidity rather than
any other effect of cooling blood (Fig. 3B). Perhaps
by reducing blood temperature there is then an
increase in tissue oxygen supply via lipid-associated
oxygen, thus explaining some of the observed benefits of lowering blood temperature during CPB.
During CPB plasma lipid-associated oxygen
increased, an unexpected observation given that
blood during this time is deliberately oxygenated to
a level resembling normal presurgical values, as
assessed electrochemically. Reasons for this discrepancy between the two forms of oxygen measurement
remain uncertain. They may reflect the effect of
haemodilution during bypass. Whereas the level of
lipid-associated oxygen is corrected for any dilution
effect by expressing units per mg of plasma protein,
conventional electrochemical measurements are not.
Alternatively, the higher level of lipid-associated
oxygen during this period may be due to a drop in
temperature which increases lipid-associated oxygen
(Fig. 3), as mentioned above. The discrepancy is
likely to be due to a combination of both haemodilution and temperature. More significant is the
observation itself. This period may be associated
with an increase in cellular debris, which may also
include an increase in transition metals. An increase
in transition metals at the same time as an increase
in lipid-associated oxygen might be expected to
enhance the deleterious processes of lipid peroxidation.
Plasma oxygen decreased by about 60% once CPB
had terminated as detected electrochemically or by
assessment of lipid-associated oxygen (Table l), suggesting an hypoxic episode. This may have been due
to a disturbance in oxygen provision by the respiratory system; this occurs as a result of microatelectasis due to accumulation of extravascular fluid in the
mediastinum and reversible alveolar collapse
[26-301. The degree to which this occurs is likely to
extend with duration of bypass. Such a possibility is
suggested by the observation that post-CPB lipidassociated oxygen was inversely related to duration
of bypass (Fig. 4A), a relationship which diminishes
with time of analysis after surgery (Fig. 4B). Conventional electrochemical and lipid-associated oxygen analysis are able to detect this short period of
anoxia (Table 1). The novel assay of lipid-associated
oxygen may well serve in the evaluation of this
potentially damaging phase of CPB. Oxygen levels
appear to have normalized 6-8 h after the end of
the operation.
To summarize, measuring lipid-associated oxygen
appears to reflect changes in plasma oxygen better
than the conventional electrochemical method. The
measurement of the partial pressure of oxygen by
conventional electrochemical means is limited at
concentration extremes. These studies have shown
that lipid-associated oxygen increased during narcosis and stages before and after CPB. Once CPB was
terminated the plasma levels of lipid-associated oxygen were significantly lower than normal for a
period of some minutes. This suggests a period of
potentially damaging hypoxia. Lipid-associated oxygen then normalized within 6-8 h of surgery.
Measurement of lipid-associated oxygen seems to
reflect subtle changes in the oxygen content of the
blood and appears to be at least as good as the conventional electrochemical measurements. The relevance of this measurement has yet to be fully
Plasma oxygen, lipid and bypass
explored. However, the elevated level of lipid-associated oxygen observed during extracorporeal blood
supply, as opposed to an unchanged oxygen tension
as assessed by conventional means, suggests the
importance of further investigation.
ACKNOWLEDGMENTS
J.V.H. would like to thank the British Heart
Foundation for his personal support.
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