Utilization of medium- and long-chain fatty acids by normal rat aorta, and the effect of Dl -carnitine on their utilization

Atherosclerosis Elsevier Publishing

Company,

UTILIZATION RAT

Amsterdam

OF MEDIUM-

AORTA,

AND

THE

345

- Printed in The Netherlands

AND LONG-CHAIN

EFFECT

FATTY

OF DL-CARNITINE

ACIDS BY NORMAL ON THEIR

UTILIZATION

S. HASHIMOTO

AND S. DAYTON

Center, Research Service and Medical Service, Wadsworth Hospital, Veterans Administration Los Angeles, Calif. 90073, and Department of Medicine, UCLA School of Medicine, Los Angeles, Calif. 90024 (U.S.A.) (Received

September

28th, 1970)

SUMMARY

Utilization

of medium-

and long-chain

effect of carnitine on their utilization

Total uptake of fatty acid, measured COs, lipid, and water-soluble

fatty acids by normal rat aorta and the

were examined

products,

was maximal

steadily with increasing chain length up to palmitate. and the degree of unsaturation

and laurate were most completely

amounting

to 99, 97 and 85%

of myristate,

ed in the same terms, was approximately

Further increase in chain length

oxidized

to COs, the fraction

palmitate,

oxidized

oleate, and linoleate, express-

54%. Added carnitine stimulated

stearate, and oleate only slightly.

of oxidation

of i4C into

and decreased

of the total uptake of fatty acids; stearate was lowest

with 33%. Extent of oxidation

completeness

with octanoate

had no effect on total uptake of fatty acid. Octanoate,

decanoate,

tion of palmitate,

in v&o.

as the sum of incorporation

Even with carnitine

of these acids did not approach

the oxidaadded the

that of the shorter chain

fatty acids. Incorporation

of 14C from radioactive

aortic lipids was investigated. aortic lipid was: phosphatide distributed

evenly

between

palmitate,

Order of incorporation > triglyceride phosphatide

oleate and linoleate into the of palmitate

> cholesteryl

and triglyceride.

and linoleate into

ester. With oleate, 14C was Incorporation

lesteryl ester was the greatest with oleate and lowest with linoleate.

into

Radioactivity

choin

aortic free fatty acid was lowest with linoleate.

This study was supported in part by a grant from The Arthur Dodd Fuller Foundation and by Research Grant HE-03734 from the National Institutes of Health. U.S. Public Health Service. Atherosclerosis,

1971, 13: 345-354

S. HASHIMOTO, S. DAYTON

346

Key words:

Carnitine

- Esterijcation

- Long-chain

fatty

acids

- Medium-chain

fatty

acids - Oxidation

INTRODUCTION Development

of atherosclerosis

cigarette

smokingr.

(FFA)sJ.

This relationship

atherosclerosis.

Common

to each

It has been suggested

The possibility

remains

is the elevation

that elevated

production

that FFA

wall to atherogenesis

the arterial

free fatty

acid

of FFA in the etiology of

plasma FFA might contribute

the study of fatty

to

by the liver?.

might in some more direct fashion predispose

and, therefore,

of fatty

acids into various

tide by arterial homogenates of approximately

the

acid metabolism

lipid components

metabolism

of arteriess.

is seemingly

Fatty

of chain

that fatty

acid contributes

acids are oxidized,

in

length

in arterial

tissues

and lysophosphaRespiratory

little to the energy

of palmitate

to influen-

however, and the rate of oxidation

on the chain length as suggested

than of palmitateii.

esterification

of cholesterol

This is suggested also by the inability

dependent

of arterial

has been studied by several investigators7,*. 1 suggests

ce glucose metabolismlo.

influence

stress and

wall is of interest.

Incorporation

octanoate

of plasma

of very low density lipoproteins

from several species has been studied 576. Esterification quotient

with diabetes,

suggests the possible importance

this disease by stimulating vascular

has been associated

This report

describes

and unsaturation

of fatty

by the greater

oxidation

a more detailed

study

acids on their

oxidation

tissues, and the effects of carnitine

of

on the

on the utilization

and

of these

acids. MATERIALS AND METHODS Incubation containing

3%

decanoate,

medium consisted albumin,

5.9 mM

[1-r%]myristate,

[I-iJC]linoleate.

Molar specific

activities

per pequiv

linoleic acid (Amersham/Searle) DL-carnitine noted).

(Calbiochem)

Bovine

albumin

was added,

water, and lyophilized

of fatty

acids except

Nuclear. mM

(except

acids and other contaminants,

Aorta from each animal was rapidly

from below the aortic arch to the diaphragm,

dialyzed stunned removed

washed with ice-cold saline, and stripped

The intima plus media was opened along the longitudinal

1971, 13: 345-354

as

with charcoal according to the proced-

Male Wistar rats weighing 250 g on the average were fasted overnight,

Atherosclerosis,

When

prior to use.

by a blow on the head, and decapitated. of fat and adventitia.

or

to the same

acid. All of the fatty was 0.10

[1-1X]-

[I-r%]oleate

approximately

from New England

its concentration

was treated

buffer (pH 7.4)

[I-r%]octanoate,

[1-r%]stearate,

were adjusted

of free fatty

phosphate

and 0.75 mM

were purchased

(Pentex)

ure of CHEN~~ for the removal against

glucose

[1-r*C]palmitate,

1.0 * 10s counts/min

value,

of 3 ml Krebs-Ringer

axis

UTILIZATION OF MEDIUM- AND LONG-CHAIN FATTY ACIDS BY NORMAL RAT AORTA

347

and transferred to ice-cold saline. Three such thoracic aortic segments were collected, blotted, and added to a flask of incubation medium (mentioned above) containing a labeled fatty acid. The flask was stoppered with a serum cap, oxygenated with the aid of hypodermic needles, and shaken for 3 h at 37°C. We have shown previously that the tissues remain metabolically active during this periodrr. Three separate experiments were done, all of which involved the same treatment of animals and the same incubation medium except as noted. Fatty

acid oxidation

to CO2

0.2 ml 5 N NaOH was added to the center well of a flask before incubation.

After 3 h, 0.2 ml 6 N HCl was added to the incubation medium and the flask was shaken for an additional 0.5 h. COs trapped by NaOH was removed and BaCOs was prepared after addition of non-radioactive NaHCOa. Incorporation

of 14C into aortic liPid and water-soluble

jwoducts

After incubation aortic segments were washed three times with fresh 0.9% saline containing 3% albumin, and left overnight in 20 ml chloroform-methanol (2: 1, v/v) containing 200 pg of non-radioactive carrier (the same fatty acid used in the incubation medium), and 200 ,ug each of cholesterol and dipalmitoyl lecithin (Mann Research)*. Control segments were dipped in the incubation medium and processed. Aortic lipid was isolated from the chloroform-methanol extract by the method of FOLCH et al.13. Steps were included in this procedure to measure water-soluble products in tissue arising from the oxidation of fatty acids. As an insurance against loss

of volatile fatty acids, the distillate was also collected during the concentration of an aortic lipid extract. The amount of radioactivity in the distillate was approximately 3 y0 of the total radioactivity of the lipid when medium-chain fatty acids were used as a substrate. Chloroform-methanol extract was separated into two phases with the addition of aqueous H&04 (1:2000) (ref. 14).The aqueous phase was back-washed with chloroform which was then added to the organic phase. Extraction procedure was tested for recovery of [ 1-r%]octanoic acid. Complete recovery in the organic phase was obtained. The organic phase was concentrated to dryness in a flash evaporator under vacuum at 60°C. This procedure was tested with [1-rJC]octanoic acid; very little radioactivity (< 1 O/J was found in the distillate. Lipid residue was assayed for radioactivity. The aqueous layer was neutralized, evaporated to dryness with ethanol and assayed for radioactivity. of [I-14C]fatty acid into lipid components After incubation with [1-rJC]palmitate, [I-rQZ]oleate or [1-r%]linoleate, aortic

Incorporation

* Non-radioactive carriers were added in order to measure radioactivitv of aortic linid comnonents. However, the radioassay procedure which we used was subsequently found to yield consistently low values for phosphatide radioactivity. Only total lipid i4C and water-soluble 14C radioactivity values are reported from this experiment. Incorporation of i4C into individual lipid fractions was restudied in the last experiment. Atherosclerosis

1971, 13: 345-354

348

S. HASHIMOTO,

S. DAYTON

segments were washed three times with ice-cold Krebs-Ringer phosphate buffer (pH 7.4) and left overnight in chloroform-methanol (2:1, v/v) containing non-radioactive carriers mentioned above. Chloroforn-methanol solution was concentrated and added to a thin plate coated with Silica Gel G (60 g/100 ml). Residual lipid, especially phosphatide which may adhere to glassis, was dissolved in chloroform-methanol (1~4, v/v) and added to the same spot on the thin-layer plate. The plate was developed in 16% diethyl ether-l o/0 acetic acid-83%

light petroleum. After the first develop-

ment the upper third of the plate was sprayed with 0.04% dichloro-fluorescein and viewed with ultraviolet light. Cholesteryl ester area was scraped into a counting vial. The plate was then developed a second time in the same direction using the same solvent mixture, in order to improve separation of the slower-moving components. After spraying and viewing with ultraviolet light the other lipid component areas were scraped into counting vials for radioactive assay. By this procedure recoveries of radioactive cholesteryl palmitate, tripalmitin, palmitic acid, cholesterol and dipalmitoyl lecithin were 94-100 %. Radioactivity

measurements were made on a Packard liquid scintillation

spectrometer. Lipid was dissolved in toluene containing 0.4 o/o 2,5-diphenyloxazole (PPO) and 0.1% 1,4-bis-2(5-phenyloxazolyl)-benzene (POPOP). Water-soluble acid salts were dissolved in toluene containing 1 y. acetic acid, 0.4% PPO and 0.1 y. POPOP. Counting efficiencies for lipid and fatty acid salt were 50% and 39%, respectively. Barium carbonate and scrapings from thin-layer plates were suspended in a thixotropic gel consisting of 43 g Hyamine 10 X (Rohm and Haas), 26 g Thixcin R (Baker Chemical Company), 3.45 g PPO, and 86 mg POPOP in 1 1 of tolueners. Counting efficiency was 42 %. Net COs radioactivity was taken as the difference between the gross counts of incubation flasks with and without aortas; this corrected simultaneously for background and for possible volatile acidic contaminants in the radioactive fatty acids. Radioactivity data were converted to “m,uatoms” of carbon 1 of the added fatty acid substrate. mpAtoms is equal to the gram atomic weight - 10-s. The rationale for expressing the data in this way is to base results on the radioactive carboxyl carbon and circumvent the uncertainty as to the fate of the remaining carbons of the fatty acid molecule. RESULTS

The effect of carnitine on the oxidation of [1-r%]palmitate is shown in Table 1. Several concentrations of DL-carnitine were used in this preliminary experiment. CO2 production was increased by 20% on an average in the presence of carnitine. In subsequent experiments 0.10 mM of carnitine was used. Utilization of medium- and long-chain fatty acids by normal rat aorta and the effect of Dr.-carnitine are shown in Table 2. The extent of stimulation of 14COs production from palmitate, stearate and oleate by camitine was small and in some experiments barely detectable; maximumstimulation was 33% in one of the stearate experAtherosclerosis,

1971, 13: 345-354

UTILIZATION

TABLE EFFECT

OF MEDIUM-

AND LONG-CHAIN

FATTY ACIDS BY NORMAL RAT AORTA

349

1 OF DL-CARNITINE

ON THE

OXIDATION

OF [l-‘4c]PALMITATE

BY RAT AORTIC

TISSUE

Incubation medium consisted of 3 ml Krebs-Ringer phosphate buffer (pH 7.4) containing 5.9 mM glucose, 3% albumin, 0.75 mM [I-14Clpalmitate (specific activity 1.06 10s counts/min per pequiv), and varying concentrations of carnitine. Tissues were shaken at 37°C under oxygen for 3 h. 14COs production in the presence of carnitine was significantly higher (P < 0.05) than the controls. DL-Carnitim

14co2

(mMl

(mpnoles/50

0 0 0 0.01 0.10 1.00

23.7 25.6 22.0 28.3 29.3 25.7

10.00

31.1

TABLE EFFECT

mg defatted dv.y tissue)

2 OF CARNITINE

ON THE

OXIDATION

OF FATTY

ACIDS

Incubation medium consisted of 3 ml Krebs-Ringer phosphate buffer (pH 7.4) containing 5.9 mM glucose, 3% albumin, 0.75 mM [1-i4C]fatty acid. When DL-carnitine was added the concentration was 0.1 mM. Tissues were shaken at 37°C under 0s for 3 h. Substrate

Octanoate Decanoate Laurate

Carnitine

0

+b

0

o+ +

Myristate

0

Palmitate

:

Stearate Oleate Linoleate

14C02 recovered (m,uatomsa of i4C/50 mg defatted dry tissue)

o+ ot i+ +

& m,u Atoms: gram atomic b With carnitine.

weight

expt. 7

expt. 2

expt. 3

447 430 221 306 64 79 58 64 28 32 18 24 30 31 35 27

276 129 298 270 63 52 50 44 22 25 10 11 20 21 24 31

312 290 162 150 46 58 27 36 27 33 13 16 25 28 32 30

. 10-s. Atherosclerosis,

1971, 13: 345-354

350

S. HASHIMOTO,

TABLE

3

INCORPORATION

Conditions

OF

14c

INTO

are the same

.Substrate

LIPID

AND

as mentioned

WATER-SOLUBLE

in Table

Decanoate

Laurate

Myristate

Palmitate

Stearate

,Oleate

Linoleate

PRODUCTS

2.

1% recovered (mpatomsb of r4C/50 mg dry &fatted tissue)

E@t.

,Octanoate

lipid

water-soluble Products

total

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2

1.2a 2.9 2.0 1.6 5.7 8.2 4.6 4.8 10.6 9.6 10.2 12.6 21.1 33.8 19.7 30.0 35.1 27.2 24.6 28.3 31.5 -

1.4 3.7 2.0 1.3 1.5 1.9 1.9 1.4 0.6 0.5 0.2 0.8 0.2 0.7 0.5 0.5 0.5 0.4 0.4 0.2 0.5

2.6 6.6 4.0 2.9 7.2 10.1 6.5 6.2 11.2 10.1 10.4 13.4 21.3 34.5 20.2 30.5 35.6 27.6 25.0 28.5 32.0 -

3 4 1 2 3 4 1 2 3 4

32.6 26.5 30.5 32.7 33.6 30.1 22.6 20.8 17.2 16.4

0 0.2 0.4 0.4 0.3 0.3 0.8 1.2 0.5 0.5

32.6 26.7 30.9 33.1 33.9 30.4 23.4 22.0 17.7 16.9

a Each value is the difference between 3 h and “zero” b mpAtoms: gram atomic weight . 10-s.

iments.

Incorporation

with increasing oleate

increase

on the incorporation

to stearate.

of 14C into lipid and water-soluble

Incorporation

of fatty

did not affect the radioactivity

there was less incorporation lipid. 1971,

values.

fatty acids into COs decreased

of the acid from octanoate

in chain length of the substrate

Atherosclerosis,

time incubation

steadily

Oxidation

of

to COa was higher than with stearate.

acids are given in Table 3. In general,

soluble products. length

of i4C from saturated

chain length

and linoleate Data

fatty

S. DAYTON

13: 345-354

little radioactivity

acids into lipids increased up to myristate.

products

from

was found in water-

Further

with progressive increases in chain

of lipid, nor did monounsaturation.

However,

of linoleic acid than of steraic or oleic acid into arterial

UTILIZATION

TABLE

OF MEDIUM-

AND

LONG-CHAIN

FATTY

ACIDS

BY NORMAL

RAT AORTA

351

4

INCORPORATION

OF

Substrate

14c

INTO

LIPID,

WATER-SOLUBLE

PRODUCTS

AND

COsa

14C recovered

(mpatomsb of 14C/ 50 mg dry defatted tissue) lipid

1.9 5.8 10.8 26.2 28.8 30.2 31.7 19.2

octanoate

Decanoate Laurate Myristate Palmitate Stearate Oleate Linoleate.

f & f f + f * +

0.7c 1.4 1.1 5.9 3.9 2.7 1.5 2.5

water-soluble products

co2

total

2.1 1.7 0.5 0.6 0.4 0.2 0.4 0.8

345 f 74 227 & 56 57* 8 455 13 26& 3 14* 3 25* 4 304 5

349

+ * * h * + * f

1.0 0.2 0.2 0.2 0.1 0.2 0.1 0.3

235 68 72 55 44 57 50

a Data from Tables 2 and 3 were combined. b mpAtoms: gram atomic weight . 10-s. c Mean * standard deviation.

TABLE

5

INCORPORATION

OF

14c

INTO

LIPID

COMPONENTS

Conditions are the same as mentioned in Table 2. 14C recovered (mpatomsa

(l-14C]Palmitate

[l-‘4C]Oleate ,[l-qLinoleate

of

14C/50 mg dry defatted tissue)

phosphatide

diglyceride and cholesterol

free fatty acid

triglyceride

cholesteryl ester

total

16.0 15.2 15.6 10.3 7.3 7.2 9.5 6.5 13.5 14.9 14.4 15.7

0.8 0.3 0.3 0.4 0.6 0.3 0.4 0.4 1.3 1.0 1.1 1.0

8.2 9.0 8.1 9.4 8.7 7.2 8.7 7.8 1.7 1.5 1.2 1.3

11.6 6.9 12.7 5.7 7.5 9.4 11.1 9.3 1.2 1.4 1.1 1.0

0.7 0.6 0.9 0.7 1.4 1.5 2.0 1.4 0.2 0.2 0.2 0.3

37.3 32.0 37.6 26.5 25.5 25.6 31.7 25.4 17.9 19.0 18.0 19.3

B m,uAtoms: gram atomic weight

. 10-s.

If the two sets of data (Tables 2 and 3) are considered

together

in Table 4 and

total uptake of a given fatty acid is taken as the sum of products measured*, seen that the medium-chain the extent

of 85-99%

* Our earlier experiment’1

fatty

of the total

acids, octanoate uptake,

through

laurate,

while the longer

yielded negligible radioactivity

in non-lipid,

chain fatty

water-insoluble

Atherosclerosis,

it can be

are oxidized

to

acids are

material.

1971, 13: 345-354

352

S. HASHIMOTO,

oxidized to the extent proportion

of 33-64 “/b.Conversely,

of the total

medium-chain

fatty

uptake fatty

The incorporation

the

oleate and linoleate into various aortic The distribution

varied with the fatty acid. With palmitate

was found in phosphatide

pally found in phosphatide

amounting

ation into sterol ester was the greatest was minimal

was confined

of 1% among

Linoleate

was distri-

radioactivity

to 75 y0 of the total radioactivity.

was princi1% incorpor-

with oleate and least with linoleate.

in triglyceride

and free fatty

these

and linoleate most of the

while with oleate the radioactivity

buted evenly between the two esterified fractions.

incorporation

for a greater

acids. Thus,

As seen in Table 5 the bulk of the radioactivity

free fatty acid and phosphatide.

lipid components

accounted

acids.

of 1% from palmitate,

lipids was investigated.

radioactivity

esterification

with longer chain fatty

acids are utilized for energy more readily and more completely

than are the long-chain

to triglyceride,

(27-36%)

S. DAYTON

Linoleate

acid fractions.

DISCUSSION

Influence compared

of carnitine

on the oxidation

to other tissueszr. Whereas

of long-chain

4-fold stimulatory

fatty

acids was small as

effects have been reported in

heart sliceszr, the effect in aortic tissue is so small as to be unconvincing. we have no information

explaining

Unequal oxidation and in mitochondria is the necessity carnitines oxidationsr.

of different fatty acids has been reported in other tissuesr7-19

crossing

fatty

oxidation

membrane

of palmitate,

carnitine.

The extent

difference

in the rates of oxidation

given for this phenomenon

acids (but not medium-chain

the mitochondrial

Our results are only partly consistent

by the stimulated

At this time

of aortic tissue.

of various organs 20. An explanation

of long-chain

before

the unique behavior

of stimulation

with this interpretation

stearate

(20%)

acids) to form acyl

to the site of fatty

and oleate

was not sufficient

of medium-chain

fatty

acid

as indicated

in the presence to account

of

for the

acids and long-chain

fatty

acids. Preferential reported

esterification

in liverr7Js,

late well with the subcellular The activating

of long chain fatty

adipose22 and intestinal distribution

enzymes for long-chain

tion of glycerophosphate for medium-chain

while the activating

of fatty acids in the incubation

acids (octanoate,

decanoate

products

-was

and laurate)

much greater

acids. The above could result from the higher concentration acids not bound

to albumin

vigorous rate of oxidation Atherosclerosis,

in the incubation

acids in the metabolic

1971, 13: 345-354

mediumss.

with

of medium-chain The significance

process has been discussed by

of medium-chain

albu-

than with long-chain

fatty

fatty

enzyme

medium containing

fatty

unbound

enzymes.

Despite this the total uptake of 14C-sum

in COs, lipid and water-soluble fatty

and esterifying

corre-

acids is found in the mitochondrionar.

min were the same in all these experiments. medium-chain

of the activating

here has been

observations

fatty acids30 and the enzyme for the esterifica-

are found in the microsomer7,

fatty

Molar concentrations of radioactivity

acids as observed

tissuesra923. These

SPECTOR~~.

fatty acids would also contribute

of The

sizably

UTILIZATION

OF MEDIUM-

AND LONG-CHAIN

FATTY ACIDS BY NORMAL RAT AORTA

353

towards greater uptake of these acids by disturbing the equilibrium between the intraarterial and extra-arterial

concentrations

of fatty acid in the direction of uptake.

Patterns of incorporation of 1% from palmitate and from linoleate into aortic lipid components were similar to those observed by others in that phosphatide fraction was the major product of these acids 5,692s. When oleate was used as a substrate, however, the radioactive label was evenly distributed between triglyceride and phosphatide. PORTMAN AND ALEXANDER3Z have shown with aortic homogenates that phosphatide synthesis occurs predominantly by the acylation of lysophosphatide. If this pathway is dominant in whole aorta as well our results reflect the selectivity of the enzyme, for the patterns of incorporation of palmitate, oleate and linoleate are qualitatively similar to that obtained by LANDS33 with liver mitochondria using lysolecithin and the CoA derivatives of these acids as substrates. Incorporation of oleate into cholesteryl ester is of particular interest because of the preferential accumulation of cholesteryl oleate in atheromata. In studies with atherosclerotic aorta, BOWYER et al .6,2’J have reported that esterifying systems discriminate in favor of oleate and that hydrolyzing systems discriminate against oleate; they have suggested that this discrimination accounts for the unique composition of cholesteryl ester in the atheroma. The data of the present experiment indicate that preferential synthesis of cholesteryl oleate is not pathological, being characteristic of normal vascular tissue. The relatively low amount of radioactivity in aortic free fatty acid when linoleate was used as substrate indicates that this acid is utilized much more rapidly following uptake by the tissue than is palmitate or oleate. ACKNOWLEDGMENTS

We acknowledge with gratitude the expert laboratory assistance of L. Berg and the help of Mrs. M. Skawienski and Miss N. Chin in preparing the manuscript.

REFERENCES MOSES, C., Atherosclerosis, Lea and Febiger, Philadelphia, 1963. FURMAN, R. H., Endocrine factors in atherosclerosis. In: F. G. SCHETTLER AND G. S. BOYD (Eds.), Atherosclerosis, Elsevier, New York, 1969, Chap. 6, p. 375. KERSHBAUM, A., S. BELLET, J. JIMENEZ AND L. J. FEINBERG, Differences in effects of cigar and cigarette smoking on free fatty acid mobilization and catecholamine excretion, J. Am. Med. Assoc., 1966, 195: 1095. STEINBERG, D., Fatty acid mobilization-mechanisms of regulation and metabolic consequences. In: G. POPJAK (Organizer) AND J. K. GRANT (Ed.), Biochemical Symposia: 24, The Control o,f Lipid Metabolism, Academic Press, New York, 1963, p. 111. STEIN, Y. AND 0. STEIN, Incorporation of fatty acids into lipids of aortic slices of rabbits, dogs, rats and baboons, J. Atheroscler. Res., 1962, 2: 400. BOWYER, D. E., A. N. HOWARD AND G. A. GRESHAM, Lipid synthesis in perfused normal and atherosclerotic aortas, Biochem. J., 1967, 103: 54. ABDULLA, Y. H., C. C. ORTON AND C. W. M. ADAMS, Cholesterol esterification by transacylation in human and experimental atheromatous lesions, J. Atheroscler. Res., 1968, 8: 967. PORTMAN, 0. W., Incorporation of fatty acids into phospholipids by cell free and subcellular fractions of squirrel monkey and rat aorta, J. Atheroscler. Res., 1967, 7: 617.

Atherosclerosis,

197 1, 13: 345-354

354

S. HASHIMOTO,

S. DAYTON

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1971, 13: 345-354