Acid-base and respiratory properties of a butrered bovine erythrocyte perfusion medium

Department oflbgediciate,M c M s s f ~ University

r Medical Cenfre,Hamillon, Our., Canada L8N 325
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Received September 4, 1985

LINDHNGER, M . I., G. J. F. HEIGENHAUSER, and N. k . JONES.1986. Acid-base and respiratory properties of a buffered bovine
erythrocyte perfusion medium. Can. J. Physiol. Phxmacol. 64: 550-555.
Current research in organ physiology often utilizes in sifu or isolated perfused tissues. We have characterized a perfusion
medium associated with excellent performance characteristics in perfused rnarnn-aalianskeletal muscle. The perfusion medium
consisting of fiebs-Henseleit buffer, bovine serum albumin, and fresh bovine erythrocytes was studied with respect to its
g a s - c q i n g relationships and its response to manipulation of acid-base state. Equilibration of the perfusion medium at base
excess of - 10, -5, 0, 5, and 10 rnmo1.k-' to humidified gas mixtures varying in their C 0 2 and O2 content was followed by
measurements of perfusate hematwrit, hemoglobin concentration, pH, CCQ?,Po2, and percent oxygen saturation. The
oxygen dissociation curve was similar to that sf mammalian bloods, having a Pso of 32 Tom ( 1 Torr = 133.3 Pa), Hill's constant n
of 2.87 2 0.15, and a B o b factor of -0.47, showing the typical Bohr shifts with respect to @02 and pH.The oxygen capacity
was calculated to be 190 d-l.-' blood. The carbon dioxide dissociation curve was also similar to that of mammaltian blood. The
in vifro nonbiearbonate buffer capacity (A[HCO,-] x A~H-')at zero base excess was -24.6 and -29.9 r n r n o l . ~ - ~ - pfor ~ - 'the
perfusate and buffer, respectively. The effects of reduced oxygen saturation on base excess and pH of the medium were
quantified. The data were used to construct an acid-base alignment diagram for the medium, which may be used to quantify the
flux of nonvolatile acid or base added to the venous effluent during tissue pedusions.

LINDBNGBW, M.I . , C.J. F. HEIGENHAUSER et N. L. JONES.1986. Acid-base and respiratory properties of a buffered bovine
erythrocyte perfusion medium. Can. J. Physiol. Pharmacol. 64: 550-555.
La recherche courante en physiologic srganique utilise souvent la perfusion de tissus in situ cs de tissus isolks. Nous avons
dCteminC lsn milieu de perfusion auquel on associe des caractCristiques de haute performance avec le muscle squelettique de
mammifbres. On a examink le milieu de perfusion compost5 d'un tampon Krebs-Henseleit, de serum-albumine et d'C~ythrascytes
frais de bovins, en ce qui a trait au transport de gaz et A la riponse au changement de l'etat acido-basique. Ap&s 19Cquilibragedu
For personal use only.

milieu de perfusion B un excks de base de - 10, - 5 , 0 , 5 et 10 m o l / L , avec des mklamges gazeux humidifids variant dam leur
teneur en C 0 2 et en 02,on dkfemina B'hCmatocsite, la concentration d'hCmepglobine, Be pH, la PCo2, le CcoZ, la Po2 et le
pourcent de saturation d'oxygbne du perfusat. La csui-be de dissociation de I'oxygkne Ctait sirnilaire a ceelle du sang de
rnmni%re, ayant uane PS0 de 32 Tom, une constante n de Kill de 2,87 k 0,15 et un facteur de Bohr de -0,47 montrant les
variations typiques par rapport au C 0 2 et au pH. La capacitk d90xyg&nedu sang etait de 190 mWL. La courbe de dissociation du
gaz carbonique h i t aussi sirnilah h celle du sang de mamifkres. La capasit6 du tampon won bicarbonate in v i m (AHCOs-/
ApH) A un excks de base zero Ctait de -24,6 et de -29,9 m o l . ~ - ' - ~ pour ~ - ' le perfusat et le tampon, respectivement. Ow a
quantifik les effets de Ba saturation rkduite d'oxygene sur I'exces de base et le pH du miiieu. On a utilise ces rksultats pour Ctablir
un diagramme d'alignement acido-basique pour le milieu, lequel peut &re utilisC pour quantifier le flux d'acide ou de base non
volatile ajoutCs B 19effluentveineux durant des perfusions de tissus.
[Traduit par la revue)

Introduction
- - . - -
perfused organ (Neely et a ~ 1975)
. and tissue (Rudeman et
d. 1971; watson1 9 8 ~ ; et al. 1985., et al. 1985)
systems have become important tools of physiological research,
allowing investigators to use a more controlled approach to
studying regulatory mechanisms than can be obtained in vkvo.
Many studies have not reported the gas-carrying properties,
buffering capacity, and ionic composition of the perfusion
mediunn employed. This tends to limit the extent to which these
studies may be extrapolated to evaluate physiological control
mechanisms existing in vivo. Precise characterization of
mammalian blood and other media used to perfuse isolated or in
sifu tissues would allow a more accurate assessment of respira-
tory and acid-base changes, since changes in any of these
variables will directly affect muscle metabolism and acid-base
regulation.
The present report describes the oxygen and carbon dioxide
carrying characteristics and the buffering capacity of a perfusate
that was found to be associated with improved physiological
characteristics in the isolated, perfused rat hindlimb electrically
stimulated to contract for 28 min (Spriet et al. 1985). The
'~uehorto whom correspondence may be sent at the following
address: Department of Medicine, Rm. 3U27, McMaster University
Medical Centre, Hamilton, Ont., Canada LWN 325.
perfusion medium consisted of fresh bovine erythrocgrtes sus-
pended in Krebs-Henseleit buffer. The studies were perfomled
at a single hemoglobin concentration, a value corresponding to
the mean rat blood hemoglobin concentration and hematoerit.
Methods
Perjusic~ramedium
The perfusate was composed of K~bs-Henseleitbuffer (Krebs and
Henseleit 1932), containing 24 M sodium bicarbonate, 50 g . ~ - '
dialyzed bovine serum albumin (Cohn fractionv), 5.6 d glucusc,
0.14 mkf free fatty acids (bound to albumin), 2.5 Mcalcium chloride,
22 pbf choline chloride, and fresh bovine erythrocytes to give a final
hemoglobin concentration ([Hb]) of 140 g.&-I. Sodium pyruvate was
added to give an initial Bactatelpyruvate ratio of 10- 15. For this study
bovine blood was collected from eight cattle of different strains, and the
erythrocytes were washed as described by Spriek et al. (1985). Briefly,
fresh bovine blood was collected directly into an ice-cold acid - citrate
- dextrose anticoagulant solution. After centrifugation the erythrocytes
were washed with 30-40 volumes of K~bs-Menseleit buffer contain-
ing 30 &bicarbonate and I 0 M glucose. Washed erythrocytes were
passed through acolumn of glass beads to remove fibrin and fibrinogen
and, prior to the addition of the buffer portion of the perfusion mediurn,
were passed through an intravenous bloo+-line filter (Abbott Labor-
atories). The buffer was initially passed through a 2 2 - ~ Hfilter
I B (Milli-
pore) then added to the erythrocytes. A preliminary study was per-
formed to determine if erythrocytes obtained from the different strains
of cattle used showed varying responses to acid or base titration. These
 LINDINGER ET AL.
TABLE1. Chemical composition of the perfusion medium compared with rat blood at 37OC
Perfusate
Pwmeter (n = 40)
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Cl-
K
' This study
ca2+
Mg2"
Glucose
Lactate
--- - -
NOTE:Vdues are mean 2 SE. All units are millimoles per litre unless otherwise noted.
perfusion media showed identical changes in measured acid-base
variables and P5@using red cells prepared from six cattle (two bulls and
four cows of three different strains),
For personal use only.
PrstocoE
Hepain was not required to prevent coagulation ~f the perfusate
because the washing procedures effectively removed the clotting fac-
tors. GIycolytic inhibitors were also not used because of their effects on
acid-base status (Nunn 1959) and their potential to displace the O2
dissociation curve (Somerkamp et al. 1961). As a result, lactate was
produced by erythrocytes at a rate of 0.24 -+ 0.04 mmo1.L-'-h-' at
37°C. To minimize the acidifying effects of lactate production, perfu-
sate samples were equilibrated to experimental conditions for no longer
than 0.5 h (see below).
The base excess of 0 m o l - L - ' for the perfusion medium was
defined at pH 7.40 and Pco2 of 40 Ton (1 Tom = 1 33.3 Pa) (Severing-
haus 1965). Aliquots (5.0 mk) of the peafusion medium or of the buffer
without red cells were transferred to chilled glass test tubes. Sodium
bicarbonate, lactic acid (molarity determined by titration with standard
NaOH), or HCI of appropriate molarity (volume of l .O m%) was added
to achieve base excess values of 0, k 5 , +- 10, and 2 2 5 mrnol-~-'.
These 6.0 mL aliquots were mixed and kept in an ice-water bath until
ready for gas equilibration. Addition of equimolar amounts of lactic
acid or HCI to the perfusion medium resulted in an identical shift in
acid-base status. Each aliquot was transferred to a 37OC temperature-
regulated tonometer (Instrument Laboratories IL 237) for 10 min and
equilibrated to humidified gas mixtures of known Po2 and Pco2 (ba-
lance nitrogen) delivered by two Wijsthoff gas mixing pumps in para-
llel. The gas flow into the tonmeter was regulated at 300 m%-min-' to
prevent dehydration of the blood. The gas mixtures were used to obtain
aliquots of the perfusate with a combination of Pcs2s of 28,40, or 60
Tom with oxygen saturations of 50,75, or 100% for each of the five
base excess levels between -10 and 10 rnmol-L-'. Only a limited
number of equlibrations were performed using base excess of k 2 5
mrno1-L;' .
Following equilibration, each aliquot was utilized for four replicates
of each measurement described below. A 708-pL sample was drawn
using a gas-aight Hamilton syringe for measurement of pH, Pco2,Po2,
percent O2 saturation, and (Hb]. An additional 200-pL sample was
taken to measure total C 0 2content ([Css2J).Additional measurements
for completion of the 0 2 and C 0 2dissociation cuwes were obtained by
equilibrating aliquots of the perfusion medium at zero base excess, pH
7.4 at the half-saturation of oxygen (P5()), and as constant Pco2 (40
Rat blood
(arterial) Reference
Lahiri 1975
Lahiri I975
Lahiri 1975
This study
Gahlenbeck et al. 1968
Schlmrb et al. 1967
Schlserb et al. 1967; this study
Gahlenbeck et a1. 1968
Schnloerb et al. 1967
This study
This study
This study
Goodman et al. 1983
This study
Goodman et al. 1983
This study
Cotlove et d.1951
Torr) or Po2 (35 Torr), while permitting the O2 and C 0 2 content,
respectively, to change. These data were used to determine the rela-
tionships between base excess, pH, and oxygen saturation, and the
nonbicxbonate buffering capacity of the perfusate and buffer. Only
those measurements made at an oxygen saturation of 95- 100% were
used to construct the perfusate acid-base alignment nomogram.
AnimaBs
To compare perfusate variables with the in vbvo condition, rat blood
acid-base, ion, and metabolite status were also determined. Mafe
Sprague-Dawley rats (354 + 1$ g, mean + SD, n = 4) were anaesthe-
tized (sodium pentobarbital, 6 mg- 100 g body wt-') and blood samples
were obtained by cardiac puncture. The samples were immediately
analyzed for whole blood and plasma acid-base state, and later for
electrolytes and substrates (Table 1).
Analytical methods
Pefusate and rat blood pH, Po2, and P C O were
~ measured at 37'C
using electrodes (Radiometer BMS3 MK2 blood microsystem coupled
to a Radiometer PHM72 MK2 digital acid-base analyzer). The pH
electrode was calibrated for each aliquot of the perfusate using preci-
sion buffers (Radiometer S 1500 and % 1510). The Pco2 and Po2 elec-
trodes were calibrated using known gas mixtures (Linde Medical Gas,
Union Carbide). Oxygen saturation and [Mb] were measured using a
Radiometer OSM2 hemoximeter calibrated with precisian Hb stan-
dads (Radiometer S218CB) and known oxygen mixtures. No spectral
differences between bovine W b (Sigma) and Radiometer Hb standards
were detected at the wavelengths (506.5 and 688.0 nm) used in the
OSM2 hemoximeter; therefore oxygen saturation characteristics were
believed to be accurately represented. C 0 2 content of whole blood and
plasma was measured using a Coming 965 C 0 2 analyzer; each sample
was bracketed with freshly made NaHC03 standards calibrated to
known solutions of WC1 to increase precision. The Corning 965 C 0 2
analyzer acidifies the sample, causing the rapid release of all C 0 2 . The
resulting changes in electrical properties of the solution are measured
and converted to a value of C 0 2 by an electrometric transducer.
Separate samples were used for the determination of the concentra-
tions of protein, glucose, lactate, free fatty acids, and ions. Protein,
glucose, lactate, and free fatty acids were measured as described by
" K' concentrations were measured using
Spriet et al. (1985). ~ a and
ion selective electrodes (Radiometer KNAl sodiudpotassium analy-
zer) calibrated using Radiometer standard solutions. Chloride was
determined by coulometric titration (Buchler-Cotlove chloridometer),
 552 CAN. B. PHYSIOL. PHARMACOL. VOL. 64, I986

and total calcium and magnesium were measured by atomic absorption TABLE2. I n vitro respiratory characteristics of bovine and rat blood and
spectrophotsmetry(Varian AA-1275). Plasma [PICO3-]was calculated the perfusion medium at 37°C
using the equation
Parameter Cattle Rat Perfusate
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where Cco2 was measured in plasma and a.(solubility coefficient for Hct 0.422 0.393
C02) was 0.8306 at 37°C (Severinghaus 1965). [wb] (g.L-8) 87-145 120-175 840
The perfusate acid-base alignment nomogram was constructed from P50 (Tom) at pH 7.4 28-32 38 32
data collected from 60 aliquots of the perfusion medium equilibrated to A 1% P~O/APH -0.49 -0.52 -0.47
95- 100%oxygen saturation, with complete analysis performed on four O2capacity ( ~ L L - ' ) 150-220 200-238 190*
samples from each aliquot. The procedure employed was essentially RBC diameter (pm) 5.9 6.0-7.5 5.9
that described by Siggaard-Andersen(1963). At pH 7.4 andPco2of 44)
Ton, the base excess of the perfusate was always within 9 2 anrno~.~-' NOTE:References: Cattle; Rms and Rsmijn 1938; Albhitton 1952; Bartels and Hams
and curve shifting to "normalize" data (Weiskopf et aI. 1983) was not 1959; Baftels e6 al. 1963; Hilpert et al. 1963; Huisman and Kitchens 1968; Rat: Albritton
1952; Bdlaad et al. 1966: Gahlenbeck et al. 1968; Gray and Steadman 1964; Hall: 1966,
required. Lahiri 1975; k c h n e r 1976.
*Calculated from [Hb] x 1.36 (Altman and Dittmer 1971).

Results
The perfusate composition (Table 1) and respiratory pmp-
erties (Table 2) closely resembled those of rat blood. Perfusate
[Mb] was within the ranges reported for both species. The B50at
pH 7.4 was in the lower range for rats but higher than that
reported for cattle. The Bohr factor (Alog BS(BIApW)and ox-
ygen capacity for the perfusate was low compared with rat
blood, but similar to bovine blood. Bovine red cell diameter was
at the lower range of reported values for the rat.
The mean O2 diss~ciationcurve of the perfusion medium
within the physiologic pH range is shown in Fig. I . Hill's n, a
For personal use only.

value which classically describes the slope of the oxygen dis-

sociation curve, was calculated to be 2.8'7 k 0.15 at pH 7.4; this
value is within the mammalian range of 2.$-3.0 (Antsnini and
Bmnofi 1971). High pH and low Pco, resulted in shifts of the
curve to the left, while low pH and high Pco2 shifted the curve to
the right. The combined B o h effect at zero base excess and
37°C. was described by the significant (P < 0.05) linear rela-
tionship: 0 20 48 60 80 100 8 20
Po, [Torr)

The cabon dioxide dissociation curve for the perfusate was
similar to bovine blood in vdlro, but to the right of the curve for
rat plasma in vivo (Fig. 2).
The reladonships between HCB3- and pH of the perfusate at
base excess values of - 10, - 5 , 0 , 5 , and 10rnrnol.~-'are given
FIG. 1. The efkct sf pH on shifting the perfusate O2 dissociation
curve at 37°C. Each point on the graph represents the mean of four
individual determinations.

slopes of these linear relations (p -

in Table 3. There was a significant correlation between the
wonbicarbonate buffer
capacity) and base excess (BE), expressed by the equations for
perfusate
and far buffer (nonerythrocyte portion):
where @ is the slope of the buffer line (in r n a n o l - ~ - ~ . p ~ - ~ ) .
The acid-base data collected from the series s f perfusate
samples from the tonometer were used to construct the acid-
base alignment diagram (Fig. 3). The errors incurred in the
construction of the alignment diagram do not exceed 2 5 % for
each scale when the perfusate acid-base status is aligned ow the
diagram.
Eflect o ~ ~ x y g ~atl~ration
en OPEbase excess
Increasing oxygen saturation produced significant linear de-
creases in both the base excess and pH of the perfusion medium,
resulting from proton release from hemoglobin. Thus the base
excess value determined for a sample of venous perfusate at an
oxygen saturation (So2)less than 95% (Po2< 75 Ton) must be
corrected to a value conesponding to that occurring at an So2of
100%to calculate the correct base excess. To calculate BE and
pH at 100% So2 from lower values of oxygen saturation, the
following equations were derived from those measurements at
perfusate oxygen saturations of 50,75 and 100%at each level of
base excess:
[5] BElm = BE, - (0.222[100%- So2]),
( r = 0.957,P < 0.05)
where BEIm is base excess at 100% So, and BE,, is the BE
measured from the alignment diagram;
-
[GI pHlm pH, -(0.004[100% - So2],
( r = 0.973, P < 8.05)
where the subscripts have the same meaning as above. The
alignment diagram thus allows the acid-base changes from
artery to vein to be converted to an equivalent amount of hy-
drogen ions added to or removed from the perfusate.
 LINDINGEW ET Ak. 553
TABLE3. The equations expressing the relationships between [HCOY]
and pH of the perfusate whole blood (WB) and buffer (P) at 37°C for
five values of base excess (BE) (millimoles per litre)

BE n Equations Y
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NOTE:The equations are in the form of a straight line; y = a 9mr where a is the y inter-
cept, ra is the slope of the noncarbonate buffer (P), y = [HCO,-] and x = pH.

2 In view of the important influences that the gas-carrying

capacity and acid-base characteristics of an organ perfusate
0 20 40 60 80 100 120 exert on the viability and function of the perfused tissue in
question, there is a surprising lack of information regarding
For personal use only.

Peo, (ferr) these properties in many previous reports. Investigations of the

RG.2. PerfusateC02 dissociation curve (heavy solid line and = 40 effects of extracellular fluid disturbances on tissue metabolism
points) compared with C 0 2dissociation curves for bovine (Bartels and quantitativedata on perfusate buffering and g a s - c q i n g
Hagans 1959) and rat (Brodie and Woodbuv 1958; Gray and Rauh capacity. This inf~n'nati~n enables XCUrate estimates of nonvo-
1958;Nichols 1958) blood at 37°C. latile acid (H+) release from tissue and organ preparations to be

BASE EXCESS

PIG.3. The acid-base alignment diagram for the perfusate at 95-180% O2 saturation, [Hb] of 140 g . ~ - ' and
, 37°C.
 554 CAN. J. PHYSIOL. PHARMACBL. VOL. 44, 1986
made. This report presents a description of the medium associ-
ated with improved performance in the rat hindlimb. The pre-
sent studies were all conducted using a single hemoglobin con-
centration approximating the mean value found in most mam-
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mals (Altman and Dittmer 1971). Since the perfusate can easily
be made up to achieve the desired hemoglobin concentration and
hematwrit, inclusion of other hemoglobin concentrations into
the alignment diagram (Siggaard-Andersew 1963; Weiskopf et
al. 1983) was not required.
Gas mmsport
Since Rudeman's (Rudeman et a1 1 97 1 ) initial character-
ization of the rat hindquarter as a model for mammalian skeletal
muscle metabolism, human erythrocytes have been used in
various gdedusican media (Rudeman et al. 1980). In early ex-
periments in our laboratory, conducted with rejuvenated time-
expired human red cells in Krebs-Henseleit buffer, a number of
problems were encountered including abnormally low P5(), ex-
cessive accumulation of lactic acid, and depletion of 2,3-
diphosphoglyceric acid (2,3-DPG), ATP, and glucose, as re-
ported also by Rudeman et al. (1988). Human red cell dimen-
sions away be up to 50% greater than rat red cells, therefore
media containing human red cells may have difficulty in perfus-
ing the capillary beds in rat skeletal muscle. This drawback does
not apply to media employing bovine red cells which are compa-
rable in size to the rat erythrocyte (Table 2). Ideally, it would be
preferable to use blood from the same species as the tissues to be
For personal use only.
perfused, a technique used by Watson (1983) for cat hindlimb
pedusisns. However, when using small animals and single-pass
perfusion systems the large amount of blood required for perfu-
sions often necessitates the use of blood from another mamma-
lian species.
The magnitude of the pH (and Pco2, not shown) induced
shifts in the O 2 dissociation curve (Fig. 1 ) was similar to that
reported for mammalian blood (Lenfant 1973). This effective
Bohr effect results from the combination of the specific separate
effects of both pH and P C Q on ~ the oxygen affinity of hemoglo-
bin (Kilnamin and Rossi-Bernardi 1973; Tyuma 1984). Ht is
clear that a decrease in pH and an increase in Pco2 will facilitate
both oxygen unloading from hemoglobin at the tissues and C 0 2
uptake by deoxyhemsglobin. These features of the perfusion
medium, combined with the normal $5(P and steep sigrnoid
shape of the O2 dissociation e w e of the perfbasion medium
(Hill's n of 2.87), suggested that the perfusate should behave
satisfactorily with respect to tissue O puptake.
Indeed, the pedusate need not be maintained hyperoxic nor
used at high flow rates to ensure adequate oxygen uptake by
isolated perfused rat hindlimb muscles during high intensity
electrical stimulation via the sciatic nerve (M. I. Lindinger and
G . J . F. Heigenhauser , unpublished observations). The prepara-
tion in current use (Spriet et al. 1986) differs slightly from that
previously described (Spriet et al. 1985) in that arterial and
venous catheters are inserted into the femoral vessels. As a
result of this refinement the total amount of tissue perfused
averages 6.5 g, with 5.3% g (or 82%) being stimulated to
contract, for a 400 g rat. With a typical resting perfusisn flow
rate of 0.3 mL.min-'=g-' the oxygen uptake averaged 0.32
p,mol.min-' .g-"erfused tissue. During the 5 min of stimula-
tion flow rates were increased to 1.5 rnLamin-'-g-' and oxygen
uptake increased to 2.58 pmol.min-'*g-' of stimulated muscle.
These flow rates are within the physiological range for these
tissues in vivo (Amstrong and Laughlin 1984) and in situ
(Mackie and Terjung 1983). Folkow and HaEicka (%968Bre-
ported similar values of muscle flow and oxygen extraction in in
situ resting and electricallystimulated soleus and gastrocnenmius
rnuse8es of the cat. These oxygen extractions are also similar to
those reported by Spriet et al. (1986) using kyperoxic perfusion
medium at the same flow rates, indicating that these flows and
n o m ~ x i coxygenation parameters were adequate for tissue
perfusion and oxygen supply. This was supported by the abs-
ence of changes in muscle concentrations of phosphocreatine,
ATP, and lactate during 20-min perfusions of resting muscle
(M. I. Lindinger and G. J. F. Heigenhauser, unpublished
observations). A recent study investigating the oxygen depend-
ence of energy metabolism in rat skeletal muscle ( I d s t r h et al.
1985)initially showed that basal metabolism could not be main-
tained at an in situ rate without the use of red cells in the
pedusate. Yet in their characterization of the oxygen depend-
ence of energy metabolism they did not use erythrocytes in the
perfusion medium and consequently had to use elevated flow
rates and high Po2 to maintain oxygen delivery at a rate at which
depletion of phosphocreatine did not occur. Clearly, perfusion
media without erythrocytes do not supply adequate O2 to the
tissues to meet metabolic demands unless unphysiologieally
high flow rates and Po2 are used.
Acid- base
The recent development of acid-base nomograms for dog
(Scott-Emuakpor et al. 1974) and swine (Weiskopf et al. 1983)
blood has shown clear species differences in acid-base charac-
teristics. The pedusate whole blood acid-base alignment dia-
gram (Fig. 3) was derived from measurements made at an
oxygen saturation of 95-100% and was designed to quantify
changes in perfusate acid-base state as it passed through the
tissues. The diagram permits rapid and accurate determination
of the entire acid-base status with the direct measurement of
only two acid-base variables, usually pH and whole blood or
plasma Ccs2 or PcB;)~. Since whole blood is used to perfuse the
tissues, changes in acid-base state and base excess of the
venous pedusate should be obtained from measurements on
whole blood.The perfusate nonbicarbonate buffer capacity over
a range of base excess values (Table 3) was within the range
reported for mammals (Altman and Bittmer 197 1;Lahiri 1975).
The perfusate buffer system responded as expected to disturb-
ances of acid-base balance induced by metabolic and respira-
tory acidosis and alkalosis, as shown by the alignment diagram
(Fig. 3).
The presence of erythrocytes in perfusion media appears to
play another role in addition to its important gas-carriage and
buffering roles. Recent studies (Watson 1983) have shown that
both protein and blood (erythrocytes plus normal electrolytes)
are required in perfusion media to prevent abnormal and exces-
sive transcapillary water exchange within the perfused tissues.
This is important in studies using perfused tissues, where chang-
ing concentrations of strong electrolytes may influence acid-
base homeostatis and, in turn, metabolism.
Acknowledgements
The authors acknowledge with appreciation the technical
assistance of Dr. M. Ganagarajah and thank Drs. D. G. McDo-
nald and C. M. Wood for the loan of equipment. This work Bias
supported by grant MA-4675 from the Medical Research Coun-
cil of Canada.
ALBRHTTON, E. C. 1852. Standard values in blood. Saunders, Phila-
delphia, PA. p. 423.
 EINDINGER ET AL. 555

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