Measuring Cellular CO2 Permeability by 18O Exchange –

Methodology and Results on Red Blood Cells 

Gerolf Gros and Volker Endeward

Zentrum PhysiologieMedizinische Hochschule Hannover

Germany

Methods Available to Measure

Membrane CO2 Permeability

● Surface pH transients in Xenopus oocytes

● Kinetics of cellular CO2 uptake recorded by intracellular pH measurement

● pH gradients in the surface region of epithelial cell layers

● Stopped flow rapid reaction spectrophotometry

● 18O exchange between CO2, HCO3- and H2O

Earlier Measurements of CO2 permeability of membranes

PCO2 of planar phospholipid bilayers from CO2 flux

measurements

0.35 cm/s (Gutknecht et al., 1977)

3.2 cm/s (Missner et al., 2008)

PCO2 of phospholipid vesicles by stopped flow

spectrophotometry

~ 10-3 cm/s (Prasad et al., 1998)

~ 10-3 cm/s (Yang et al., 2000)

Can the kinetics of CO2 and O2 uptake by red cells be

reliably measured by stopped flow techniques?

t1/2 of CO2 uptake by human red cells: 13 ms

(Holland and Forster, 1975)

continuous-flow rapid reaction apparatus

t1/2 of CO2 uptake by red cells by theory: ~ 12 ms(Endeward et al., 2008)

t1/2 of O2 uptake by human red cells: ~ 80 ms

(Vandegriff and Olson, 1984)

Determining Membrane Permeabilities of CO2 and HCO3-

by the 18O Exchange Technique

Has been applied to

Isolated cells in suspension: red blood cells, MDCK and tsA201 cells

Phospholipid vesicles in suspension

Intact colon epithelium

HC18O16O2- + H+

H216O + C18O16O

H218O + C16O2

Mass spectrometer

2/3

1/3

time (s)

0 100 200 300 400 500

[ C

18O

16O

] (

µM

)

4

6

8

10

12

14

datafit

red cells

CA

HC18O16O2- + H+

H216O + C18O16O

H218O + C16O2

Cell

H C18O16O2- + H+

Mass spectrometer

CAH2

18O + C16O2

H216O + C18O16O

PHCO3-

PCO2

PH2O

2/3

1/3

1/3

2/3

time (s)

0 100 200 300 400 500

[ C1

8 O1

6 O ]

( µ

M )

4

6

8

10

12

14

datafit

red cells

CA

Magnetic stirrer

Mass spectrometer

Stirring bar

pH meter Blood cells

Teflon membrane

mass46/mass 44

Water 37°C

HC 18 O 16 O 2- + H +

H 216 O + C 18 O 16 O

H 218 O + C 16 O 2

Red Cell

H C 18 O 16 O 2- + H +

Mass spectrometer

CAH 2

18 O + C 16 O 2

H 216 O + C 18 O 16 O

P HCO3 -

P CO2

P H2O

2/ 3

1/ 3

1/3

2/3

Time (s)

0 100 200 300 400 500

[ C

18O

16O

] (

µM

)

4

6

8

10

12

14

datafit

red cells

CA

)()()()(

)()(

/tOOHCtOOHC

H

H

v1

vaPtOOHCHA

K

k

OH

tOHCOtOOCAk

dt

tOOHCdin2

1618ex2

1618

in

exHCOex2

1618exex

1

u

2

ex18

22ex

1618exu

ex21618

3PHCO3-

)()()()()(

/tOOCtOOCaPtOOHCHA

K3

k2tOOCAk

dt

tOOCdex

1618in

1618COin2

1618inin

1

uin

1618inu

in1618

2 PCO2AinAin

)()()()(

)()(

/tOOHCtOOHC

H

HaPtOOHCHA

K

k

OH

tOHCOtOOCAk

dt

tOOHCdin2

1618ex2

1618

in

exHCOin2

1618inin

1

u

2

in18

22in

1618inu

in21618

3PHCO3-Ain Ain

)()()()(

)(/

tOHtOHv1

vaPtOH

OH

COAktOOHCHA

K3

k1

dt

tOHdex

182in

182OHex

182

2

2exuex2

1618exex

1

uex18

22

PH2O

)()()()(

)(/

tOHtOHaPtOHOH

COAktOOHCHA

K3

k1

dt

tOHdex

182in

182OHin

182

2

2inuin2

1618inin

1

uin18

22

PH2O

)()()()()(

/tOOCtOOC

v1

vaPtOOHCHA

K3

k2tOOCAk

dt

tOOCdex

1618in

1618COex2

1618exex

1

uex

1618exu

ex1618

2

PCO2

Fig.3

Time (s)

0 100 200 300 400 500

[ C

18O

16O

] (

µM

)

4

6

8

10

12

14

datafit

red cells

CA

Why can we observe fast processes on such a slow time scale,

allowing us to follow these processes by mass spectrometry?

Time (s)

0 100 200 300 400 500

[ C

18O

16O

] (

µM

)

4

6

8

10

12

14

datafit

red cells

CA

time (s)

0 1000 2000 3000

[C1

8 O1

6 O] M

5e-6

6e-6

7e-6

8e-6

9e-6

1e-5

time (s)

0 10 20 30 40 50

[CO

2] M

0,0012

0,0014

0,0016

0,0018

0,0020

0,0022

0,0024

0,0026

0,0028

0,0030

CO2 + H2O ↔ HCO3- + H+

↔ H216O + C18O16O

↔ H218O + C16O2

HC18O16O2- + H+

t1/2 = 5 s t1/2 = 250 s

Kinetics of CO2 hydration reaction vs. that of 18O exchange

2/3

1/3

Time (s)

0 100 200 300 400 500

[ C

18O

16O

] (

µM

)

4

6

8

10

12

14

datafit

red cells

CA

It was shown here that a time course of the decay of [C18O16O] that is

measurable by mass spectrometry, is observed when the volume fraction of

human red cells is extremely small, i.e. 2 x 10-4. Raising this volume fraction

by a factor of 10, to 0.002, renders the signal already too fast compared to

the time resolution of the mass spectrometer in combination with the inlet

system.

It is concluded that the process of 18O exchange can be slowed down by

orders of magnitude, because it is possible to use extremely small amounts

of red cells and still obtain a well-defined and clear signal.

Also for this reason, the 18O exchange technique allows us to observe

fast processes such as the uptake of CO2 by red cells on a very slow time

scale.

How well are PCO2 and PHCO3- defined by the

experimental curves of decay of [C18O16O] ?

It was shown here that a well-defined minimum exists for both PHCO3- and

PCO2 in the sum of squares of deviations between the experimental data

of [C18O16O] and those obtained from the best-fit calculation.

When PHCO3- and PCO2 are varied over a wide range of values, clearly only

one well-defined minimum is apparent and no local minima whatsoever

are visible.

t (s)

0 100 200 300 400

0,1

1

A = 4

uncatalysed

[C18

O16

O]

– [C

18O

16O

]

8

t (s)

0 100 200 300 400

0,1

1

PHCO3- = 0.0015 cm/s

PCO2 = 0.0015 cm/s

PCO2 = 0.015 cm/s

PCO2 = 0.15 cm/s

Ae = 4

[C18

O16

O]

– [C

18O

16O

]

8

Sensitivity of calculated PCO2 to parameter values

a +

10

%

a -

10 %

K'1

+ 1

%

K'1

- 1

%

Ai +

5 %

Ai -

5 %

pH

e +

0.0

1

pH

e -

0.01

pH

i + 0

.1

pH

i - 0

.1

PH

2O +

10

%

PH

2O -

10

%

Per

cen

t ch

ang

e in

PC

O2

-40

-30

-20

-10

0

10

20

30

To what extent do unstirred layers around cells

affect the permeability determinations?

thickness of unstirred layer ~

kinematic viscosity x √cell diameter ℓ

Landau LD and Lifschitz EM (1991): Hydrodynamics

solutionCO

UL

memCOappCO DPPe

,,, 222

11

0 1 2 3 4 50

5

10

15

20

Viscosity (10-6 m2/s)

1/P

CO

2 (s

/cm

)

saline

Papp in saline (cm/s)

PMcm/s)

in salineµm)

37 °C 0.12 0.16 0.5

~

CO2 Permeability of Normal and Deficient

Human Red Blood Cells

no

inh

ibit

or

1 m

M p

CM

BS

2 m

M p

CM

BS

3 m

M p

CM

BS

10 µ

M D

IDS

100

µM

DID

S

PC

O2 (

cm/s

)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

controlColton Null

*

* *

** * *

*

PCO2 of control and AQP1 deficient (Colton null) human red blood cells

Endeward et al., FASEB J, 2006

PC

O2

(cm

/s)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

controlColton Null

*

* *

** * *

*

Endeward et al.,

2006

Time (s)

0 50 100 150 200 250 300

[ C

18O

16O

] (

µM

)

6

8

10

12

14

16

18

20

Rh Nullcontrol

Red Cells

cont

rol

cont

rol +

10

µM D

IDS

Rh Nul

l

Rh Nul

l + 1

0 µM

DID

S

PC

O2 (

cm

/s)

0.00

0.05

0.10

0.15

0.20

0.25

***

#

***

§

PCO2 of control and Rhesus null human red blood cells

Endeward et al.,

FASEB J, 2008

Human Red Blood Cell

cont

rol

cont

rol +

10

µM D

IDS

Rh

Nul

lR

h N

ull +

10

µM D

IDS

AQ

P1

Nul

l

AQ

P1

Nul

l + 1

0 µM

DID

S

PC

O2

(cm

/s)

0.00

0.05

0.10

0.15

0.20

0.25

*

*

*

##

Endeward et al.,

2006, 2008

Applying the 18O technique to measure the

CO2 permeability of the apical membrane of

intact colon epithelium

thermostattedwater jacket

pH electrode teflon plug

CO2

tomass spectrometer

stirring bar

magnetic stirrer

colon mucosaonteflon cylinder

teflon membraneonsintered glass disc

Mass Spectrometer

Epithelial Cell

HC18O16O2- + H+

PHCO3-

2/3

1/3

H216 O + C18O16O

H218O + C16O2

PCO2

HC18O16O2- + H+

H216 O + C18O16O

H218O + C16O2

1/3

2/3

PH2O

CA

Intact Proximal EpitheliumApical Side

time ( s )

0 100 200 300 400 5001.5

1.7

1.9

2.1

2.3Epithelium + extracellular CA inhibitor

CA

[C1

8O

16O

] (1

0- 5

M)

PCO2 ± SD

(cm/s)

PHCO3-

(cm/s )

Ain n

Intact

Proximal

Colon

0.0015 0.0007

6.3 10-4

4.0 10-4

41 000 40

Intact

Distal

Colon

0.00077

0.00021

0.87 10-4

0.56 10-4

900 23

CO2 and HCO3- Permeability of the Apical Membrane

of Intact Guinea Pig Colon

Endeward & Gros, 2005

Conclusions

The 18O exchange technique follows the decay of 18O-labelled CO2

in the extracellular fluid by mass spectrometry

This is possible because this decay is 1.000-10.000 times slower than netCO2 uptake by cells or vesicles

The system of differential equations describing this process yields values ofPCO2 and PHCO3- from well defined minima of a fitting procedure

PCO2 values can be determined over a range of 3-4 orders of magnitude

Parameters critical für calculation of PCO2 and PHCO3- are intracellular CAactivity and extracellular pH, both of which are carefully controlled

Unstirred layers affect the results by no more than ~ 20%

The method is applicable to suspensions of isolated cells or vesicles and tointact epithelia