BIOCHEMICAL MEDICINE 20, 279-283 (1978)

LETTER TO THE EDITOR

The Effects of 0, Concentration and Albumin on Respiration and Aerobic Glycolysis in Rabbit

Aortic Intima-Medial.’

A recent report by A. D. Winegrad e? al. (9) has suggested that the low rates of oxygen uptake and high rates of aerobic glycolysis, widely ob- served in aortic intima-media studied in vitro (l-8) are not intrinsic to the tissue but result artifactually from methods of tissue preparation. The authors state that their preparation which shows preservation of intact endothelium exhibits much lower relative degradation of glucose via the glycolytic pathway and much higher oxygen uptake. Conditions of prepa- ration were: sedation of rabbits in order to avoid agonal struggle before killing by decapitation, 6% albumin (purified by dialysis) added to the Krebs-Ringer bicarbonate (KRB)-glucose buffer used for tissue collec- tion and incubation, and unopened aorta stripped of adventitia from the outside (FTIM .382 mm in thickness) as opposed to our technique of stripping a thin layer (0.189 mm) of intima-media (IIM) from the lumenal side (3). Additionally, 0, uptake was measured using 95% O,-5% CO, as the saturating gas phase with the YSI monitor instead of 95% air-5% CO, as recommended for use with this instrument. We have reexamined the effects of these conditions and also of age difference of the rabbits on oxygen uptake and glycolysis.

Table 1 illustrates the results of an experiment utilizing FTIM prepared according to methods described in (9). Two groups of animals, “young” and “mature” were studied. 0, uptake and lactic acid production (IO) were measured on replicate samples (75-100 mg) in duplicate cuvettes containing KRB (3 ml) with either air-5% CO, or 95% O,-5% CO, as the saturating gas phase. There are no significant differences in oxygen up- take between aortas of young and mature animals with either air or 95% 0, as the gas phase. When blank values, representing oxygen escape from the medium saturated with 95% 0, are subtracted before calculation of

’ Supported by NIH Grant HL 14177. 2 Preliminary report presented at Second Annual Hugh Lofland Conference on Arterial

Wall Metabolism, May 1977.

279 OOO6-2944/78/0202-0279$02.00/O

Copyright @ 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.

280 LETTER TO THE EDITOR

tissue oxygen uptake, the values are not different than in air saturated medium and not different than our previously reported values (5). As can be seen from Table I, the blank value is a large percentage of the total observed oxygen uptake in the 95% 0, saturated medium. The unmodified YSI incubation cell is not compatible with the use of gas mixtures greatly different from ambient.

Lactate values are also not significantly different in aortic IM from young and mature animals with either gas phase or between gas phases.

Table 2 reports the effects of the addition of albumin to the incubation medium and of stripping technique on 0, uptake and lactate production. The effect of 95% 0, saturation on tissue 0, uptake was not evaluated, for reasons given above, but was evaluated on tissue lactate production. The addition of 6% albumin to the incubation medium greatly increased 0, uptake in both types of tissue, but to a significantly greater degree in IIM than in FTIM. If one makes the assumption that 0, enters the tissue by free diffusion according to the Warburg relationship (1 I). then the thickness of neither type of tissue is such as to limit the observed rate of oxygen uptake. However, there is some evidence that a steep oxygen gradient does obtain in some kinds of vascular tissue (12-14) and thus 0, penetration into deeper layers of the tissue may not be a simple diffusion process.

Lactate production of FTIM, in either air saturated or 95% O,-saturated medium was not significantly different in the presence or absence of albumin (Table 2 vs Table 1). However, lactic acid production was signifi- cantly higher in IIM than in FTIM whether measured in air or 95% O,-saturated medium. Since both 0, uptake and glycolysis went up pro- portionately in the former, it is possible that the full thickness IM contains tissue which is less active than inner IM alone.

Table 3 shows a comparison of data previously published by us (5) with that of A. D. Morrison er al. (9). The apparent differences between the two sets of data are in oxygen uptake, which, as we have demonstrated, is due in large part to the presence of albumin in the medium, and in [ 14C]glucose oxidized to 14C0,. The latter investigators used uniformly labeled glucose in their experiments, while we used [6-14C]glucose; thus, the 14C0, they report is the sum of pentose shunt and Krebs cycle activity. If pentose shunt activity in rabbit aorta is of the same order of magnitude relative to Krebs cycle as in swine aorta (5), then 65% of their reported activity from [U-14C]glucose is not used directly for energy production. Thus, the presence of albumin in the medium greatly in- creases 0, uptake but does not increase the contribution of glucose to the energy supply of the FTIM over that reported for IIM tissue either via Krebs cycle or glycolysis.

Oxygen uptake can be taken as a rough measure of Krebs cycle activ-

TABL

E I

0,

UPTA

KE”

AND

LAC?

‘A

r~

PROD

UCTI

ONS

OF

RABB

IT

AORT

K FT

IM’

IN

AIR

AND

95%

0,

”

Youn

g vs

M

ature

An

imal

s

Youn

g (7

) M

ature

(7

)

Body

we

ight

(8)

1462

f

112

3350

rt

188

Air-5

%

CO,

0,

upta

ke

Lact

ate

98.4

4.

56

‘- 19

rt

1.1

95.9

5.

33

k 17

zt

1.0

0,

upta

ke,

appa

rent

304

r 46

21

8 -+

30

95%

o,-

5%

co,

0,

upta

ke

Diffu

sion

blank

(-

diffu

sion

blank

) (%

of

to

tal)

110

64.5

-t

2-7

2 3.

9 86

.7

61

rt 24

lr:

8.

3

Lact

ate

6.38

t

1.3

5.77

4

0.5

0 0,

Up

take

, pl/

g/hr

. *

Fmol/

g/hr

.

w c

Aorta

st

rippe

d of

ad

vent

itia

from

ou

tsid

e.

3c

d Re

sults

ar

e ex

pres

sed

as

mea

ns

+ SE

. Fi

gure

s in

pa

rent

hese

s ar

e nu

mbe

rs

of

anim

als.

In

cuba

tion

med

ium:

KRB

+ 6

mM

gluc

ose,

pH

7.

4.

TABL

E 2

EFFE

CT

OF

ALB~

~MIN

AN

D ST

RIPP

ING

TE

CHNI

QUE

(FTI

M

vs

IIM)

ON

O2

UPTA

KE

AND

GLYC

OLYS

IS

OF

RABB

IT

AORT

A IN

CUBA

TED

IN

AIR-

~%

CO,

AND

95%

0,

-Y%

CO

,”

Body

we

ight

Air-S

%

CO,

95%

o,-

5%

co,

w 0,

up

take

” La

ctat

e’

Lact

ate

FTIM

(9

) 26

07

160

4.67

4.

00

t 89

k

14

i

k 0.

7 t

0.8

p <

0.05

p

< 0.

025

p cc

0.0

25

IIM

(5)

2890

20

8 7.

15

7.05

1

rt 48

z!z

20

ir 0.

8 rt:

0.

9

I’ In

cuba

tion

med

ium:

KRB

+ 6

mM

gluc

ose

i 6%

al

bum

in,

pH

7.4.

h

&‘g/

hr.

( pm

ollg

ihr.

Lact

ate

was

dete

rmine

d on

on

ly th

ree

pairs

of

ao

rtas.

282 LETTER TO THE EDITOR

TABLE 3 COMPARISON OF 0, UPTAKE AND GLUCOSE DEGRADATION BY RABBIT AORTIC IM INCUBA-

TION IN KRB-GLUCOSE IN THE ABSENCE AND PRESENCE OF ALBUMIN

Percentage 0, [“C]Glucose ( uptake accounted

Tissue Albumin 0, uptake to ‘TO for by glucose Lactate preparation concentration (pi/g/hi) (hmol/g/h:) oxidation (pmol/g/hr)

A” 0 96 2 12 0.099 + 0.02 14 3.40 k 0.5 B” 6% 210 + 7 0.177 2 0.013 II 3.44 2 0.24

” From E. S. Morrison et nl. (5). e From A. D. Morrison et rrl. (9). ’ [6 - ‘“C]Glucose used in A. “CO, produced would be from Krebs cycle activity.

[U-‘4C]Glucose used in B. i4C0, produced would be sum of pentose shunt and Krebs cycle activity.

ity, but does not permit the assumption that the major substrate for this activity is glucose. In our previously reported study (5), the mole ratio of glucose degradation via the glycolytic pathway to that via Krebs cycle activity was 13 in normal rabbit aorta. In Morrison and.Winegrad’s study (9), when r4C0, was not corrected for pentose shunt activity this ratio was 10 and where corrected > 20. In swine and monkeys we have found an even higher proportion of glucose degradation to proceed via aerobic glycolysis. Significantly, when one calculates the amount of oxygen which must be utilized to burn the amount of glucose degraded to CO, in the three species discussed, it amounts to only six to 15% of the observed oxygen uptake in both studies. Since there is no apparent difference in amounts of glucose degraded by FTIM by either pathway when the incubation medium contains albumin, but the oxygen uptake is doubled, it must be concluded that oxidation of some other substrate must be af- fected. Even this rate of O2 uptake, of course, is small as compared to that of liver and kidney.

REFERENCES I. Kirk, J. E.. Effersoe. P. G., and Chiang, S. P. J. Grrontol. 9, IO (1954). 2. Lehninger, A. L., in “The Arterial Wall,” (A. I. Lansing, Ed.) Williams & Wilkins,

Baltimore, 1959. 3. Scott. R. F.. Morrison, E. S.. and Kroms. M., J. Atheroscler. Res. 9, 5 (1969). 4. Scott, R. F., Morrison, E. S., and Kroms, M., Amer. J. Physiol. 1363 (1970). 5. Morrison, E. S., Scott, R. F.. Kroms, M.. and Frick, J., Atherosclerosis 16, 175 ( 1972). 6. Lille, R. D.. and Chobanian, A. V., J. C/in. Invest. 48 (Part I), S2a (1969) [Abstract 1661. 7. Peterson, J. W., and Paul, Richard J., Biochim. Biophys. Acta 357, 167 (1974). 8. Arnqvist, H. J.. and Lundholm, L., Atherosclerosis 25, 245 (1976). 9. Morrison, A. D., Berwick, L., Orci, L., and Winegrad. A. I., J. C/in. Invest. 57, 650

(1976). IO. Gutman, I., and Wahlefeld. A. W., in “Methods of Enzymatic Analysis.” (H. U.

Bergmeyer and K. Gawehn. Eds.), 2nd ed.. Vol. 3. pp. 1464-1468. 1974.

LETTER TO THE EDITOR 283

II. DeLuca. H. F.. and Cohen. P. P.. i~t “Manometric Techniques,” (W. W. Umbreit. R. H. Burris. and J. F. Stauffer, Eds.), 4th ed., pp. 116-l 17. 1964.

12. Pittman, R. N., and Duling, B. R. Microvasc. Res. 6, 202 (1973).

13. Fay, R., in “Tissue Hypoxia and Ischemia,” (M. Reivich. R. F. Coburn, S. Lahini and B. Chance, Eds.), Plenum Press, New York, 1977.

14. Jurrus, E. R., and Weiss, H. S., Fed. Proc. 36, 4708. (1977) [Abstract].

E. S. MORRISON J. FRICK

M. KROMS Depurtment of Pathology

Albany Medical College

Albany, Nen, York 12208

Received October 27, 1977