Role of the liver in the metabolism of exogenous cholesterol esters

ARCHIVES

Role

OF

BIOCHEMISTRY

of the

Liver

AND

BIOPHYSICS

in the

the Veterans .4dministralion School CJ~ Medicine,

541-553

Metabolism

LEON SWELL?, From

106,

(1964)

of Exogenous

nI. D.LAW

ARTD

December

Esters1

C.R.TREADWELL

Center, Martinsburg, West Virginia, The George Washington Ilniversity, Received

Cholesterol

and the Department of Biochemistry, Washington, D. C.

3, 1963

Rats were fed a test meal containing cholesterol, no fatty acids, or different fatty acids. A control group received a test meal with only a tracer amount of cholesterol4-C14. Substantial amounts of cholesterol esters accumulated in the liver in 24 hours. The degree of accumulation was greatest when unsaturated fatty acids were fed. The cholesterol ester which accumulated to the greatest extent, irrespective of the fatty acid fed, was cholesterol oleate. The fatty acid composition of the serum cholesterol esters was essentially unchanged by cholesterol feeding. The cholesterol-Cl1 ester pattern in the liver at 24 hours was similar in all groups irrespective of the fatty acid fed and showed a general shift toward Cl”-monounsaturated esters. In all groups, the principal cholesterol-C’4 ester found in the blood at the end of 24 hours was cholesterol arachidonate. The specific activity data on the free and individual cholesterol esters, of the liver indicate that a major portion of the incoming cholesterol esters undergoes: transferase reactions with fatty acid donors to form other cholesterol esters. The, specific activity of the serum cholesterol esters did not exceed the specific activity of the corresponding liver cholesterol esters. The results of the present study suggest that intestinal cholesterol esters are rapidly removed by the liver. A portion of these esters undergoes hydrolysis to free cholesterol while another portion appesrs to participate in a series of transferase react.ions with fatty acid donors to form other cholesterol esters. A major ester formed in this fashion is cholesterol arachidonate. which is then released into the blood in association with the lipoproteins. Several cated the

earlier liver,

lar, oleic acid (5, 11). There is also evidence that there may be additional enzymatic pathways for the formation of cholesterol esters. Washed rat liver microsomes have been shown to esterify free cholesterol in the presence of ATE’ and CoA (7, 8). A transferase type reaction has also been shown to occur in serum (3). That enzyme catalyzes the esterification of free choIesteroE with the fatty acid in the P-position of lecithin to give a cholesterol ester and a lysolecithin. The fate of cholesterol esters in vivo has also received some attention. Recent studies of Goodman (4) and I~ossow et al. (12) have shown that lymph chylomicron cholesterol esters are rabidly removed from the circulation by the liver and undergo substantial hydrolysis in that tissue. However, no evidence has been presented to date

reports (l-4) have impliintestine, adrenal glands, the forlnation of the blood

and the blood in and tissue cholesterol esters. A number of in vitro studies (5-10) have shown that several tissues (pancreas, intestinal mucosa, liver, adrenal, and blood) contain enzymatic systems for synthesizing cholesterol esters. The enzymic system present in the pancreas and intestinal mucosa has been characterized as a cholesterol e&erase type enzyme (5, 6). This enzyme shows a marked preference for esterification of cholesterol with unsaturated fatty acids and, in particu* This work was supported in part by IJSPHS ,(;rants HE-01897 and HE-04374. * Present address: Lipid Research Laboratory, Veterans Administration Hospital and Medical College of l’irginis, Richmond, Virginia 541

542

SWELL,

LAW AND

regarding any additional reactions which these lymph cholesterol esters may undergo in the liver and perhaps other tissues. The information obtained on the metabolism of blood and tissue cholesterol esters has been concerned with the esterified fraction as a unit; this fraction contains a number of individual cholesterol esters which may have more than one origin and metabolic function. Klein and Martin (13) were the first to show that the major liver cholesterol esters have different turnover rates which may be a result of the fatty acid specificity of the esterification enzymes or differences in the rate of movement of cholesterol esters in and out of the liver. Processes involving the selective retention or release of certain cholesterol esters by the liver must occur since the esters of the liver and blood have different fatty acid compositions (14-16). Further evidence for a selective retention and releaseof cholesterol esters by the liver was obtained in a recent study by Roheim et al. (17). These investigators found that the blood cholesterol esters of D < 1.019 had a fatty acid composition very similar to the esters of the liver. The cholesterol esters of the high-density serum lipoproteins (D = 1.063-1.21) consisted predominantly of polyunsaturated esters whereas the liver fraction contained mostly saturated and monounsaturated esters. The purpose of the present study was to obtain information on the metabolism of individual exogenous cholesterol esters. Data (both chemical and Cl”) are presented on the role of the liver in the uptake and release into the serum of the individual exogenous cholesterol esters after feeding cholesterol-4-Cl4 with and without different fatty acids. The results suggest that the liver plays an important role in the selective retention and release of cholesterol esters; changes in the metabolism of endogenous cholesterol esters were also observed.

TREADWELL group consisted of 20 animals. The animals were fasted overnight, and 5 groups received, by intubation without anesthesia, 3 ml. of an aqueous emulsion containing 41.1 mg. of cholesterol-4-Cl4 (2.5 PC), 50 mg. blood albumin, 292 mg. of fatty acid, 279 mg. sodium taurocholate and 159 mg. glucose. A control group received 3 ml. of an aqueous emulsion containing a tracer amount of cholesterol-4-V (2.5 PC), 50 mg. blood albumin, and 150 mg. glucose. The cholesterol-4-C’* was obtained from the Nuclear-Chicago Corporation, the blood albumin from Fisher Scientific Company, and the purified fatty acids (98-99%) from the Hormel Foundation. After administration of the test meal, 5 animals in each group were sacrificed at intervals of 2, 4, 6, and 24 hours. At each time blood was obtained from the abdominal aorta, and the liver was removed and weighed. The serum and liver were extracted with 2:l chloroform-methanol as previously described (18).

hlETH0~s The free and esterified cholesterol fractions of the serum and liver extracts were separated on silicic acid columns (19). Separation and recovery of the free and esterified cholesterol fractions were routinely checked with cholesterol-4-CY and known cholesterol-4-Cl4 esters. The free and esterified cholesterol fractions were assayed for cholesterol by the method of Sperry and Webb (20) The C’4-activity of the free and ester sterol fractions was determined on the digitonides in a liquid scintillation system as previously described (21). A portion of the cholesterol ester fraction was analyzed for fatty acid composition by gasliquid chromatography (15). Another portion of the cholesterol ester fraction was separated into 4 major cholesterol ester classes (saturated, monoand arachidonate) on unsaturated, linoleate, silicic acid columns by a modification of the method of Klein and Janssen (22, 23). The CY-activity of the cholesterol ester classes isolated from serum and liver was then determined. Separation of the cholesterol ester classes was checked with known pure cholesterol-4-C’” esters (cholesterol-4-Cl”-palmitate cholesterol-4-Cla-oleate, cholesterol-4-C14-linolea\e, and cholesterol-4-C14arachidonate) prepared as described elsewhere (24). RESULTS

EXPERIMENTAL TREATMENT

OF

ANIMALS

AND

TISSUES

The experimental animals were male rats, weighing 180-225 g., of the Carworth strain which had been maintained on Purina pellet chow. The animals were divided into 6 groups and each

CHOLESTEROL AND

LEVELS LIVER

IN

SERUM

The serum and liver free and cholesterol ester levels are shown in Table I. The groups receiving the cholesterol meals with oleic,

CHOLESTEROL

ESTER TABLE

CHOLESTEROL

LEVELS

IN Live?

Time after feeding (hours)

Free

SERUM

AND

cholesterol

Tracer 1.7 1.6 1.5 1.6

values

FEEDING

f & f f

cholesterol-4-C’4 0.5 11.3 0.5 10.7 0.4 10.3 0.6 11.0

2 4 6 24

13.4 13.3 13.0 13.0

f I.6 zt 1.8 + 2.0 * 1.7

2 4 6 23

13.1 14.5 14.2 14.9

f 2.0 & 1.9 k 3.0 + 2.4

2 4 G 24

13.4 15.0 13.G 14.7

f zt + Ik

2.0 3.1 2.5 2.9

2 4 G 24

14.4 14.3 14.3 15.0

z!z zt + +

2.3 1.9 2.4 2.1

2 4 G 24

16.3 10.9 17.3 15.5

* It It *

3.5 2.2 2.0 1.7

Cholesterol-.-C” 2.2 * 1.0 2.8 + 1.1 5.2 f 1.8 8.7 h 2.5

2 4 6 24

14.4 15.5 15.8 17.3

f + f f

0.8 1.0 1.2 1.2

Cholesterol-d-P4 1.7 + 0.G 3.0 zk 0.8 3.9 It 1.0 12.0 XL 2.4

Cholesterol-4-F4; 1.9 f 0.5 2.8 + 0.9 2.3 f 1.1 5.1 * 1.0 Cholesterol-4-C14 2.2 z!z 0.6 2.2 + 0.8 3.7 i 1.1 5.0 * 1.5 Cholesterol-4-C’” 2.1 + 0.5 4.7 f 0.9 4.4 zk 1.8 8.1 z!z 1.1

SEVERAL Serum”

7” Ester

(total)

Free

TEST

MEALS

cholesterol Ester -

(mgi%) 7% Ester (total)

---

(control) & 3.8 f 4.1 f 3.5 & 4.3

12.4 13.7 14.1 14.4

41.7 38.0 49.7

78.1 75.3 72.9 77.5

no fatty acid 12.7 & 4.1 16.2 It 5.4 13.9 f 4.6 25.5 zk 5.1

11.4 12.3 12.3 12.0

40.6 37.1 39.9 38.8

78.1 75.1 76.4 7G.4

13.1 13.9 15.6 13.9

41.4 39.8 48.1 49.0

7G.0 74.1 75.5 77.9

15.9 13.2 16.8 16.6

54.5 62.1 56.0 70.4

77.4 82.1 76.9 80.9

12.1 12.9 15.6 14.9

41.4 39.1 48.1 49.0

77.3 75.2 75.5 76.7

15.4 15.1 16.5 14.8

46.3 45.3 51.2 54.9

75.0 75.0 75.6 78.8

+ pulmitic 14.1 + 12.8 * 21.4 k 25.4 +

+ oleic acid 12.7 zk 4.1 24.7 zk 5.8 23.5 zt 6.0 35.1 f 4.2 f

linoleic 11.9 zk 14.2 & 23.1 f 36.0 f

44.2

acid 5.1 4.6 6.1 5.3

acid 3.8 5.1 5.7 4.9

+ arachidonic 11.3 zk 4.0 16.2 f 5.1 19.8 f 5.3 41.0 * 4.5

acid

per whole liver to show absolute changes in ester level; average 6.6 & 0.4 g. Each liver value represents the average of 5 animals i on serum represent a pool of 5 animals. was

or arachidonic acids had a 4-7-fold in the liver cholesterol ester level at the end of 24 hours when compared to the control group. Significant increases were present in the liver cholesterol esters in the above groups in as little as 4 hours after feeding the test meals; at 24 hours the percentage of esterified cholesterol was 3040 % as compared to 11% in the control group. The groups receiving the cholesterol test meal with no fatty acid or palmitic acid also showed an increase in the liver cholesterol esters at the 6- and 24-hour

linoleic, increase

AFTER

(mg./liver)

~1Calculated groups

I

LIVER

Ester

543

METABOLISM

liver weight for all standard deviation;

periods. The increase at 24 hours (about 3-fold) was much less than occurred in the other cholesterol-fed groups; the percentage of esterified was 20-27 %. The liver free fraction of the several cholesterol-fed groups showed a slight increase at the end of 24 hours. The serum cholesterol ester fraction of the groups receiving the cholesterol meals showed virtually no change except for the group receiving the oleic acid in which there was a 25% increase at 24 hours. The serum ester to total ratio was constant in the groups at the various times and was

544

SWELL,

LAW AND TREADWELL

approximately 77 %. There was no change in the serum free cholesterol fraction of the several groups. The levels of the individual cholesterol esters in serum and liver are shown in Table II. There were increases in the levels of saturated, monounsaturated, and linoleate esters in the livers of the cholesterol-fed groups. The increase was greatest at the end of 24 hours. The arachidonate ester showed a slight increase in the livers of the animals receiving the arachidonic, oleic, and linoleic acids. In all of these groups the greatest elevation was in the monounsaturated liver cholesterol esters. For

example, there was a greater increase in the liver monounsaturated esters than in the saturated esters in the animals fed palmitic acid. The level of the individual serum cholesterol esters of the groups fed the test meals with cholesterol, no fatty acid, and palmitic and linoleic acids showed virtually no change. The group fed the oleic acidsupplemented test meal showed an increase in the monounsaturated serum esters (300%) and a 40% increase in the linoleate ester at the end of 24 hours. The group fed the arachidonic acid test meal had a notable increase in the level of monounsaturated esters (180%) after 24 hours.

TABLE LEVEL

OF INDIVIDUAL

CHOLESTEROL Liver

Time after feeding (hours)

Saturated

0.7 0.6 0.6 0.6

0.5 0.5 0.5 0.5

2 4 6 24

0.8 1.2 0.8 1.4

0.6 0.8 0.9 2.1

2 4 6 24

0.8 0.7 1.1 1.4

0.7 0.8 1.5 2.1

2 4 6 24

0.7 1.2 1.3

1.9

0.8 2.4 2.2 3.7

2 4 6 24

0.6 0.7 1.2 2.0

0.7 0.8 1.9 3.8

2 4 6 24

0.6 0.9 1.2 2.6

0.4 1.1 1.4 5.5

from

data

esters*

MOllOUIb saturated

2 4 6 24

a Calculated

ESTERS

on fatty

AND LIVER

AFTER FEEDING

(mg.)

Tracer 0.3 0.3 0.3 0.3

SEVERAL

Serum Esters”

Linoleate

Arachidonate

Saturated

cholesterol-.J-C~4 0.2 0.2 0.2 0.2

Cholesterol-.$-P; 0.3 0.5 0.4 1.2

no fatty

+ palmitic 0.3 0.2 0.4 0.3

Cholesterol-.-C’4 0.3 0.8 0.7 2.1

+ oleic 0.2 0.2 0.2 0.4

Cholesterol-d-Cl4 0.7 1.1 1.8 2.3 Cholesterol-404 0.3 0.6 0.8 2.7 composition

+ linoleic 0.2 0.2 0.3 0.5 +

arachidonic 0.4 0.5 0.5 1.2 and levels

MOIIOUUsaturated

TEST

MEALS

(mg.) Linoleate

Arachidonate

0.6 0.5 0.5 0.7

0.4 0.4 0.4 0.4

0.7 0.6 0.7 0.6

1.7 1.9 1.7 2.1

0.6 0.6 0.7 0.5

0.4 0.5 0.6 0.4

0.6 0.6 0.8 0.6

1.7 1.7 1.2 1.8

acid 0.5 0.6 0.6 0.5

0.4 0.4 0.5 0.4

0.5 0.6 0.6 0.5

1.7 1.7 1.9 2.1

0.5 0.6 0.5 0.7

0.5 0.8 0.9 1.2

0.6 0.8 0.6 1.0

1.9 1.7 1.7 1.7

acid 0.7 0.5 0.5 0.5

0.6 0.5 0.5 0.4

1.0 0.8 0.8 0.5

2.3 2.5 1.8 1.8

0.5 0.7 0.7 0.9

0.7 0.7 0.7 0.7

2.2 1.6 2.1 2.0

acid

0.2 0.3 0.2 0.4

Cholesterol-&P 0.4 0.5 0.7 1.2

acid

II

OF SERUM

acid

acid 0.5 0.6 0.5 0.7 of cholesterol

esters

(Tables

I and III).

CHOLESTEROL

ESTER

METABOLISM

in the percentages of the fatty acids in the liver cholesterol ester fraction in as short a In Table III is shown the fatty acid time as 4 hours in the groups fed the chospectrum of the serum and liver cholesterol lesterol test meals. At the end of 24 hours all ester fractions. The individual cholesterol the cholesterol-fed groups had from 41 to ester fatty acids were combined into the 46% monounsaturated fatty acids in the major fatty acid classesfor comparison with ester fraction. At the same time, there was the data on the cholesterol-4-U4 ester a drop in the percentage of saturated and classesisolated by the modified Klein and arachidonic acids. The percentage of linoleic .Janssen procedure (22, 23) as shown in acid in the liver cholesterol esters showed Table II. There were substantial changes very little change in the several groups FATTY ACID COMPOSITION OF SERUM AND LIVER CHOLESTEROL ESTERS

TABLE F.~~.TY

ACID

III

COMPOSITION OF SERUM AND LIVER CHOLESTEROL AFTER FEEDING SEVERAL TEST MEALS % Total

Time feedi&

after (hours)

Liver

fattv

Serum Arachidonate

Tracer 2 4 6 24

38.4 38.1 41.6 37.3

31.0 32.1 30.0 30.7

2 4 6 24

40.6 44.1 33.6 26.6

30.9 27.1 38.9 41.2

2 4 6 24

38.0 32.4 30.5 27.2

30.9 38.7 39.6 42.5

2 4 6 24

34.9 25.6 29.4 23.6

39.7 51.3 51.0 45.6

2 4 6 24

29.2 25.6 23.7 23.3

30.3 29.2 35.8 44.1

2 4 6 24

37.4 29.6 30.1 21.5

22.8 35.2 37.0 45.9

acids

esters”

Saturated

Cholesterol-4-04 18.3 12.3 18.7 11.1 17.6 10.8 20.0 12.0

Vholesterol-Q-C”; 16.7 17.4 18.7 23.5 Cholesterol-.-P4 19.2 24.5 20.3 23.6 Cholesterol-Q-C14 10.3 17.4 15.1 25.8 Cholesterol-4-C’1 31.2 36.9 34.3 26.5 Cholesterol-.&G4 18.6 19.4 20.3 22.6

Q Values represent analysis on a pool a Fatty acid composition determined classes for comparison with composition

ESTERS

Saturated

Monounsaturated

estersa Linoleate

Arachidonate

(control) 18.1 14.9 15.8 18.6

12.0 11.9 12.8 10.2

19.7 17.7 19.6 16.4

50.2 55.5 51.8 54.e

acid 17.2 16.5 19.9 14.9

12.4 13.7 18.0 12.9

18.7 19.1 24.3 17.3

51.7 50.7 37.8 54.9

+ palmitic 11.9 10.4 9.6 6.7

acid 17.5 17.6 17.1 14.1

11.8 11.5 12.5 11.3

16.5 18.2 15.8 14.8

54.2 52.7 54.6 59.8

+ oleic 8.0 4.7 4.5 5.0

acid 14.2 14.7 13.6 15.5

13.2 20.8 24.0 25.4

18.2 21.0 16.5 21.3

54.4 43.5 45.9 37.8

+ linoleic 9.3 8.3 6.2 6.1

acid 14.5 12.4 13.2 15.3

12.9 10.8 14.0 13.1

21.5 19.1 21.9 15.0

51.1 57.7 50.9 56.6

13.3 19.2 16.4 21.7

17.8 18.4 17.4 15.8

56.1 45.3 53.1 47.0

no fatty 11.8 11.4 8.8 8.7

+ arachidonic 21.2 15.8 12.6 10.0

acid 12.8 17.1 13.1 15.5

of liver and serum from 5 animals. by gas-liquid chromatography; fatty acids of cholesberol-Cl4 esters (Table V).

grouped

into

major

546

SWELL,

LAW AND

except for the one receiving the linoleic acid-supplemented test meal. That group had a high percentage of linoleic acid particularly at the early times (2 and 4 hours). The fatty acid composition of the cholesterol ester fraction in the liver at the end of 24 hours was not influenced by the type of fatty acid fed. The serum cholesterol ester fatty acid composition of all the groups showed very little change as a result, of feeding cholesterol except for the group fed the oleic acidsupplemented meal. That group showed a doubling of the percentage of oleic acid in the serum cholesterol ester fraction at the end of 24 hours. Of particular interest is the observation that the percentage of arachidonic acid in the serum cholesterol esters remained remarkably constant throughout the 24-hour period and averaged from 50 to 55% of the total cholesterol ester fatty acids. The polyunsaturated fatty acids accounted for approximately 70% of the total serum cholesterol ester fatty acids. Comparison of the serum and liver cholesterol esters indicates that the feeding of the test meal substantially altered the pattern of the liver cholesterol ester fatty acids without any appreciable change in the serum cholesterol ester fraction. Also, it is shown that the serum and liver cholesterol esters have a widely differing fatty acid spectra. The liver cholesterol esters contain primarily saturated and monounsaturated fatty acids, and the serum esters consist predominantly of polyunsaturated fatty acids. RECOVERY OF ADMINISTERED CHOLESTEROL-4-C'4

The percent recovery of fed cholesterol4-C?* is shown in Table IV. The recovery of cholesterol-4-C?* in the liver of the cholesterol-fed groups at the end of 2 hours was on the order of 1-2.5 %. However, of that portion recovered, from 40 to 70 % was in the esterified form. The groups fed the oleic acid-, linoleic acid-, or arachidonic acidsupplemented mealshad a greater proportion of the cholesterol-C’* in the esterified form (at 2 hours) than the other cholesterol-fed groups. The percent recovery of cholesterol-

TREADWELL

Cl* as ester declined after 2 hours in all groups but was still substantially above the group receiving the tracer meal of cholesterol-4-C’*. At, the end of 2 hours a substantial amount, of the recovered cholesterolCl* in liver was in the esterified form in the group receiving the tracer meal of cholesterol-4-C14. It then declined rapidly and was the sameduring the 6-24-hour interval. Recovery, in the liver, of the administered cholesterol-4-C’* was greatest in the groups fed the unsaturated fatty acid-supplemented test meals. The group fed the cholesterol meal with no fatty acid had the lowest percentage of recovered cholesterol-4-C’* (5.7%) and the group fed the palmitic acid test meal had only 11.5 % of the cholesterol4-C’* present in the liver at 24 hours. The group receiving the tracer meal of cholesterol-4-C?* had only 7.8 % of the administered cholesterol-4-C’* in the liver at the end of 24 hours. The percentage of the administered cholesterol-4-Cl4 recovered in the serum of the cholesterol-fed groups was much lower than that recovered in the liver of these corresponding groups at the various times. The highest recovery of cholesterol-4-C’* in the serum was observed in the groups fed the unsaturated fatty acids. The percentage of total cholesterol-C?* in the esterified form in the serum showed virtually no change throughout the 24-hour period and ranged from 70 to 80%. COMPOSITION OF LIVER AND SERUM Cl*-CHOLESTEROL ESTERS

The above data have provided some information regarding the over-all handling of cholesterol esters in serum and liver following the feeding of several cholesterol test meals with and without different fatty acids. Data on the cholesterol-4-C’* ester composition (Table V) provide more information on the handling of the newly synthesized cholesterol esters. These data were obtained by isolating the individual cholesterol ester classes on silicic acid columns and determining the Cl*-activity associated with those esters in serum and liver. In the groups fed the cholesterol test meals with palmitic, oleic, linoleic, and

CHOLESTEROL

ESTER TABLE

RECOVERY

OF

IV

FED

cHOLESTEROL-4-c’4

70 Fed Time feedingh

after (hours)

547

METABOLISM

cholesterol-4-C”

recovered

Livera

Free

Ester

SeMll’d

~

Total

Total I

Tracer 2 4 6 24 ~~.-

2.5 , 3.8 ’ 3.0 6.8 ~~.

f f * +

0.5 0.6 0.5 0.7 ~~~~~

~ 1.5 ~ 0.8 1 0.5 ~ 1.0

f f + f

cholesterol-~-O4

0.5 0.3 0.2 0.4 , ~-~

4.0 4.4 3.5 7.8

f f zk l

0.8 0.7 0.5 1.0

Cholesterol-4-04; 2 4 6 24

~ 0.G 2.5 ~ 3.9 1 3.9

zt z!z zt *

0.2 0.5 0.8 0.8

0.4 1.1 1.3 1.7

rt Ilt -1. &

0.2 0.4 0.5 0.6

1.0 3.6 5.2 5.6

0.6 3.9 5.4 7.5

f + f f

0.2 0.6 0.8 1.0

0.7 2.0 2.6 4.0

f + + +

! g

;

;Lj

10.1

+

2.5

I

0.8 5.0 6.8 12.0

+ + + f

0.9 5.7 11.3 8.9

f f f f

0.4 0.7 2.1 1.7

1.6 3.0 6.7 8.1

=k + f f

0.3 1.2 2.0 3.0

I 1.2 8.9 13.8 ‘22.1

0.6 1.4 2.0 2.4

’ 2.5 9.3 18.0 17.0

Cholesterol-4-C” 2 4 F 24

1.2 2.8 4.1 10.9

f f + +

z!x f ff

6.0 5.4 G.4 3.7

acid

40.0 30.1 25.0 30.3 ~

palmitic

+ oleic .____ zk f + +

0.3 0.6 1.1 2.1

1.7 6.5 11.0 24.6

0.4 1.5 3.0 4.9

7.0 8.1 9.2 10. 1 .~~

+

arachidonic

f f f zk

0.4 1.2 2.3 3.8

0.04 0.15 0.27 0.23

) 0.1-l 0.56 , 0.96 ~ 0.81

0.18 0.71 1.23 1.04

77.8 78.9 78.0 77.9

0.05 0.19 0.43 0.50

0.18 0.42 0.82 ~ 1.82

0.23 0.61 1.25 2.32

78.3 68.9 65.6 78.4

0.03 0.23 0.44 0.86

0.11 1.10 1.43 3.31

0.14 1.33 1.87 4.17

78.6 82.7 76.5 79.4

~ 0.06 0.32 0.57 0.42

0.33 0.70 1.78 1.G5

1 0.39 1.02 2.35 2.17

84.6 68.7 75.7 76.0

1 0.06 0.36 0.59 0.93

’ 0.27 1.27 1.70 3.43

0.33 1.63 2.29 4.36

81.8 77.9 74.2 78.7

acid

+ linoleic 0.9 1.9 3.4 3.1

72.7 72.1 65.6 76.2

acid

/ G6.7 56.2 49.3 54.3

f f zk f

0.55 0.68 0.61 2.56

-

f zk f 4~

0.2 0.5 0.7 1.0

Cholesterol-,$-C’” 2 4 6 24

0.5 1.0 1.1 1.5 +

Cholesterol-.-O4

-__.2 4 6 24

* + f f

37.5 18.2 14.3 12.8

no fatty

Cholesterol-4-C’4 2 4 6 24

Tt,;s,; I

64.0 38.7 37.2 47.6

70.6 43.1 37.3 44.3

+ rt f +

10.1’ 9.0’ 7.4 11.1

acid zk ziz zk f

9.6 7.2 6.4 8.4

acid rk f & f

8.4 6.9 7.5 9.5

0 Calculated per whole liver and per total serum volume; the total serum volume was estimated from body weight and hematocrit. Each value for liver represents the average of 5 animals + standard deviation; values on serum represent a pool of 5 animals. * Each animal in the cholesterol-fed groups received, in addition to other constituents described in text, a test meal containing an average of 41.1 & 2.5 mg. cholesterol-4-P (2.5 pc, average specific activity 63,000 cpm./mg.); an additional group received a test meal with a tracer (60 y) amount of cholesterol-4-C’4 (2.5 PC).

548

SWELL,

LAW

AND

arachidonic acids the CWcholesterol ester composition was related to the corresponding fatty acid fed; e.g., when palmitic acid was included in the test meal, at the end of 2 hours 76.2 % of the liver C4-esters were saturated. Also, when oleic and linoleic acids were fed, the monounsaturated and linoleate esters accounted for about 60% of the cholesterol-U4 esters at the end of 2 hours. In the case of arachidonic acid, the percentage of cholesterol arachidonate did not exceed 280/G. When no fatty acid was administered or a tracer dose of cholesterol-

4-Cl4 was fed, the C14-cholesterol esters were predominantly saturated and monounsaturated. Following the 2-hour period, there was a progressive shift in the liver cholesterol-CL4 ester composition of all the groups. The major changes were an increased percentage of CL4-monounsaturated esters and a smaller increase in the percentage of cholesterol-C4 arachidonate. At the end of 24 hours, the liver cholesterol-Cl4 esters of all the groups were similar in composition. Comparison of these data (Table V) with those shown in Table III shows that, with

TABLE COMPOSITION

OF SERUM

AND

LIVER

TREADWELL

V

CHOLESTEROL-4-Ct4

ESTERS % Total

Time feedine

after (hours)

Liver Saturated

unsyEaied

Tracer 22.1 20.7 24.0 21.4

32.6 32.2 32.2 35.5

2 4 6 24

42.8 39.3 36.7 21.6

35.4 36.1 37.6 45.6

Cholesterol-.&P; 17.9 19.2 19.1 20.5

2 4 6 24

76.2 64.2 65.8 30.8

12.8 20.9 18.5 39.0

Cholesterol-.J-P4 8.8 11.5 11.4 18.8

2 4 6 24

22.4 21.6 22.6 22.1

60.5 60.2 59.8 51.3

2 4 6 24

20.7 26.7 26.5 24.3

17.3 25.9 30.2 39.4

2 4 6 24

27.9 27.8 25.2 19.4

18.9 26.2 31.7 40.2

Cholesterol-Q-C14 25.6 25.4 25.6 24.6

TEST

MEALS

C’“-esters

Saturated

unEtzoa;ed

Linoleate

Arachidonate

35.8 28.5 17.0 10.7

32.8 20.2 14.6 8.0

17.4 14.6 19.6 17.5

14.0 36.7 48.8 63.8

acid 41.4 37.8 33.8 37.8

29.2 29.2 28.5 15.0

10.4 15.8 15.9 12.6

19.0 17.2 21.8 54.6

+ palmitic 2.2 3.4 4.3 11.4

acid 61.0 44.2 26.6 11.2

18.5 15.4 16.2 11.9

11.0 14.8 13.6 15.8

9.5 25.6 43.6 61.1

+ oleic 3.8 3.2 4.8 5.9

acid 26.8 27.6 27.1 13.6

54.0 44.9 42.3 25.1

12.3 14.1 16.1 20.3

6.9 13.4 14.5 41.0

+ linoleic 2.3 3.2 6.8 9.1

acid 20.9 20.7 21.7 12.6

20.5 16.7 19.8 13.2

35.6 39.8 35.1 17.6

23.0 22.8 23.4 56.6

18.0 22.2 20.2 20.0

23.9 21.8 18.5 15.3

28.9 30.0 39.8 49.7

$2:;

cholesterol-4-C14 5.5 6.8 10.5 23.1

Cholesterol-.J-C14 13.3 15.0 12.8 20.7 Cholesterol-4-C14 59.7 44.2 36.5 27.2

SEVERAL

Serum C~~-esters*

Linaleate

39.8 40.3 33.3 20.0

analysis classes

FEEDING

04.ester8

2 4 6 24

0 Values represent b Cholesterol ester

AFTER

no fatty 3.9 5.4 6.6 12.3

+ arachidonic 27.6 20.6 18.5 15.8

acid 29.2 26.0 21.5 15.0

on a pool of liver and serum of 5 animals. were separated on silicic acid columns and

their

CL’-activity

determined.

CHOLESTEROL

ESTER

minor exceptions, the composition of the liver V-esters and the fatty acid composition of the cholesterol esters were similar. This suggests that at the end of 24 hours the exogenous cholesterol-U4 had become equilibrated into the various cholesterol ester pools in the liver. The serum C14-ester composition is also shown in Table V. At the end of 2 hours the composition of the liver cholesterol-CL4 esters was similar to the serum C14-ester composition. This similarity at the early time suggests t,hat the newly absorbed cholesterolLC’4 esters were present in the blood and entering the liver, e.g., when oleic acid was fed the serum, C14-monounsaturated esters at 2 hours accounted for 54% of the total serum C14-esters and the percentage of P4-monounsaturated esters in the liver at that same time (2 hours) was 60%. With time, there was a marked shift in the pattern of serum CQsters. In all of the groups the principal shift was toward an increase in the percentage of cholesterol-4-C14-arachidonate. At the end of 24 hours, cholesterol-4-CY4arachidonate accounted for approximately 50% of the total C14-esters. At the same TABLE LEVEL

OF INDIVIDUAL LIVER

549

METABOLISM

time, there were concomitant drops in the percentages of the other C14-esters. At the end of the 24-hour period, the C14-ester composition of all of the groups was remarkably similar except for an increased percentage of monounsaturated fatty acids in the group fed the oleic acid test meal. Also, the serum CY4-ester composition and cholesterol ester fatty acid pattern were similar at the end of 24 hours, indicating that essentially all of the serum esters had become equilibrated. Comparison of the liver and serum CY4-ester patterns at the end of 24 hours substantiates what was pointed out earlier with respect to the fatty acid composition of the cholesterol esters, namely, that the liver and serum CYesters are not in equilibrium. The major C14-esters of the liver are monounsaturated and saturated whereas in the serum fraction P-polyunsaturated esters predominate. EXDOGEXOUS CHOLESTEHOL

A&-D

EXOGENOUS LEVELS IN

LIVER

The endogenous and exogenous cholesterol ester levels in the liver are shown in VI

ENDOGENOCS AND EXOGENOUS CHOLESTEROL AFTER FEEDING SEVERAL TEST MEALS Cholesterol Saturated

Monounsaturated

esters=

ESTERS

(mg.) Linoleate

Arachidonate

Test meal fed

Liver

Tracer cholestjerol-4-W (control) Cholesterol-4-U4; no fatty acid Cholesterol-4-Cl4 + palmitic acid Cholesterol-4-C14 + oleic acid Cholesterol-4-C’4 + linoleic acid Cholesterol-4-C14 + arachidonic acid

(24 hours) 0.5

0.G

OF

0.3

0.2

1.2

0.2

1.7

0.4

1.0

0.2

0.3

0.1

0.9

0.5

1.4

0.7

0.9

0.3

0.1

0.2

0.9

1.0

1.3

2.4

1.2

1.0

0.1

0.3

1.2

0.9

2.4

1.4

1.3

1.0

0.2

0.3

1.7

0.9

3.7

1.8

l.G

1.1

0.5

0.7

a Values represent an average of 5 animals for liver; exogenous esters calculated from covered in liver ester fraction and specific activity of fed cholesterol-4-Cl4; endogenous calculated from total est’er level minus exogenous esters.

total cpm reesters were

550

SWELL,

LAW AND

Table VI. There were substantial amounts of both endogenousand exogenoussaturated, monounsaturated, and linoleate esters present in the liver 24 hours after feeding the cholesterol test meals. The striking feature of the data is that the increase in endogenous esters was greater than the amount of exogenousesters, e.g., in the caseof the group fed the linoleic acid-supplemented diet there was a greater increase in endogenous cholesterol oleate than in exogenous cholesterol oleate. The major exception to this observation was the group fed the oleic acid-supplemented diet, where the exogenous cholesterol oleate exceeded the endogenous

TREADWELL

fraction. The endogenous esters accounted for more than one-half of the total cholesterol esters present in the liver of the several groups. The endogenous level of the arachidonate ester showed essentially no change except for the group receiving the arachidonic acid-supplemented diet. However, the exogenous cholesterol arachidonate accounted for a large proportion of the total liver cholesterol arachidonate. SPECIFIC ACTIVITY SERUM

TABLE

VII

AND CHOLESTEROL ESTERS OF SERUM FEEDING SEVERAL TEST MEALS

feeding

Liver free and cholesterol esters (cpm./mg.

(hours)

Saturated

$$$$

Linoleate

Tracer 405 215 174 269

X 102)

Serum

AND LIVER

free and cholesterol

esters

AFTER

(cpm./mg.

X 102)

Arachidonate

cholesterol-J-C’4 149 125 124 436

2 4 6 24

71 101 88 198

348 206 102 135

349 197 135 291

2 4 6 24

11 43 70 67

60 85 158 70

Cholesterol-&C’4; 65 63 123 106 140 145 95 75

18 45 108 123

2 4 6 24

11 72 117 138

154 464 388 283

Cholesterol-.-C14 32 35 128 111 84 102 185 160

2 4 6 24

9 94 166 230

67 232 311 362

160 326 473 434

2 4 6 24

14 89 167 146

135 332 369 246

Cholesterol-4-C’4 111 370 274 383 278 351 211 243

2 4 6 24

9 63 114 207

137 229 232 215

Cholesterol-4-C’” 86 238 344 309

Cholesterol-.&P4 152 252 181 321 236 348 208 259

LIVER AND ESTERS

The specific activities of the free and cholesterol esters in the serum and liver are

SPECIFIC ACTIVITIES OF FREE

Time after

OF

CHOLESTEROL

49 77 66 210

88 102 49 109

122 89 52 149

40 44 45 204

12 35 43 222

acid 13 39 68 65

25 95 126 74

24 89 117 72

6 35 49 45

4 14 43 61

+ palmitic 14 76 81 341

acid 15 53 94 127

53 85 91 104

24 46 75 137

10 27 51 140

3 16 47 134

f 49 189 426 450

acid 7 70 124 214

16 134 205 162

34 155 183 185

56 48 102 179

11 22 33 203

+ linoleic 50 121 364 354

acid 12 51 115 137

27 69 205 107

29 65 179 132

31 86 201 154

8 16 53 131

+ arachidonic 241 322 406 376

acid 12 75 113 216

42 140 184 207

25 106 138 198

25 108 119 208

9 61 84 228

no fatty

oleic

CHOLESTEROL

ESTER

shown in Table VII. The specific activity of the free cholesterol in the serum and liver approached the same value in the 6-24.hour interval in all of the groups. At the end of 24 hours the specific activity was highest for the cholesterol ester corresponding to the fatty acid incorporated into the test meal. For example, in the case of the oleic acidsupplemented meal, the specific activity of the monounsaturated cholesterol esters far exceeded that of any of the other liver cholesterol esters. In all the groups, cholesterol arachidonate had the lowest specific activity at the end of 2 hours. The peak specific activity of the individual cholesterol esters was reached at varying times beyond 2 hours, depending on the fatty acid incorporated into the test meal. For example, in the case of the oleic acid-supplemented test meal the peak was reached in 6-24 hours. It is also shown that the individual liver cholesterol esters had differing specific activities at the various time intervals, and even at the end of 24 hours the individual cholesterol esters had not come into equilibrium. The arachidonate ester had the highest specific activity of the various esters at the end of 24 hours. The most striking feature of the data is that the individual liver cholesterol esters had a specific activity equal to or, in most cases, higher at the different time intervals than the fret fraction. In the case of cholesterol arachidonate, the specific activity of that ester exceeded the free at the end of 24 hours by a factor of at least 2. The serum free cholesterol fraction had a specific activity equal to or greater than any of the esters at the different time intervals in all of the groups with some minor exceptions. Cholesterol arachidonate had a lower specific activity than the other esters at the early time interval (2 hours) and did not reach its maximum until 24 hours had elapsed.At the end of 24 hours the individual cholesterol esters approached the same specific activity. The individual serum cholesterol esters of the several groups had a lower specific activity than the corresponding esters in the liver. This is particularly true in the case of cholesterol arachidonate, where the specific activity of this ester in

METABOLISM

551

the liver was considerably higher than in the serum. DISCUSSION

In agreement with the earlier findings of Goodman (4) and Lossow et al. (la), it is shown that cholesterol esters derived from the intestine are very rapidly removed from the circulation. Thus, at the early time (2 hours) the amount of administered cholesterol-CY in the liver was much greater than was present in the serum, and the specific activity of the individual liver cholesterol esters at that time great.ly exceeded the specific activity of the esters in the serum. This indicates that the absorbed cholesterol esters are preferentially removed by the liver. Analytical data (2, 4, 16) obtained on the liver, serum, and lymph cholesterol esters of the rat have shown that the cholesterol esters of those tissues have differing fatty acid compositions. Also, recent data (17, 25) have indicated that the cholesterol esters of the very low-density and high-density serum lipoproteins have differing fatty acid compositions. All of these data, taken together, suggest that the liver modifies the incoming absorbed cholesterol esters before they are released back into the blood. In the present study, the composition of the cholesterol-C4 esters present in the liver and serum at 2 hours was practically identical. Since at the later times the composition of the cholesterol-P esters in the serum and liver was different, the liver must have introduced substantial changes in the cholesterol-O4 ester pattern; the predominant C4-ester released back into the blood, probably as a component of the high-density lipoproteins, is cholesterol arachidonatc. The cholesterolMY ester composition and the fatty acid composition of the cholesterol esters in the serum and liver were virtually identical at the end of 24 hours, which suggests that the various cholesterol ester pools had become equilibrated in the serum and liver. The feeding of the different fatty acids with cholesterol did not influence the release of cholesterol-U4 arachidonate into the blood. This was true even though substantial

552

SWELL,

LAW AND

accumulation of cholesterol esters of the saturated and monounsaturated types occurred in the liver. The mechanismsinvolved in the synthesis of the serum high-density lipoproteins by the liver must be highly specific since cholesterol arachidonate is the principal ester released into the serum irrespective of the fatty acid fed or when cholesterol esters of the saturated and monounsaturated types accumulate in the liver. There is evidence that there are several metabolic pathways involved in the synthesis and transformation of cholesterol esters. The chylomicron cholesterol esters taken up by the liver undergo hydrolysis to an appreciable extent (26). The present data also confirm these findings and indicate that in the animals given the tracer meal of cholesterol-4-Cl4 almost all of the cholesterol-C14 recovered in the liver after the 2-hour period is present in the free form. In the animals fed a cholesterol load there was also a substantial amount of the recovered cholesterolCl4 present in the liver in the free form, but this was after the 2-hour period. However, due to the accumulation of cholesterol esters in the liver, the proportion of the total cholesterol recovered as free cholesterol at 24 hours was only about 60%. Another possible metabolic route for the cholesterol esters entering the liver is suggested from the present study, namely, that the cholesterol esters undergo a seriesof transferase reactions with fatty acid donors to form other cholesterol esters. These newly formed cholesterol esters can then be incorporated into the different cholesterol ester pools of the liver and serum lipoproteins. The specific activities of the individual liver cholesterol esters, in comparison with the free fraction, clearly show that a major portion of the cholesterol esters present in the liver could not have arisen by esterification of free cholesterol. At 24 hours, sufficient time elapsed for the exogenous cholesterol entering the liver to come into equilibrium with the various cholesterol ester pools. The much higher specific activity of the liver cholesterol arachidonate at the end of the 24-hour period clearly indicates that this cholesterol ester could not have arisen from

TREADWELL

the free fraction. The varying specific activities of the individual liver cholesterol esters are in agreement with the earlier findings of Klein and Martin (13) and points up the heterogeneous character of the liver cholesterol esters. They have different turnover rates which might result from differences in fatty acid specificity in the esterification process or differences in the individual fatty acid pools associated with the esterification reaction. Thus, the available information indicates that there are at least several pathways for the synthesis of cholesterol esters. One pathway involves the esterification of free cholesterol with fatty acid derived from a donor (21). Another pathway involves a transferase reaction between free cholesterol and the ,&fatty acids of lecithin (3), and still another pathway, as shown in the present report, may involve the reaction of cholesterol esters with a lipid donor in the liver to form other cholesterol esters. Thus, the pattern of cholesterol esters synthesized by the liver and released into the blood may be the resultant of several synthetic pathways. Several points can be made regarding the possible origin of the serum cholesterol esters. The specific activities of the individual serum cholesterol esters were always lower than the corresponding cholesterol esters in the liver. The slow rise in the specific activity of the serum cholesterol arachidonate is probably due to the necessary time lapse for the turnover of that serum ester, but it is highly significant that at the end of 24 hours 50-60% of the total cholesterol-Cl4 estersin the serum was present as cholesterol arachidonate. At the same time (24 hours), the liver cholesterol arachidonate had the highest specific activity of the different cholesterol esters. These data taken together suggest that the liver plays a central role in furnishing the cholesterol esters as part of the various lipoprotein fractions. Klein and Martin (27) previously reported an expansion of the liver cholesterol oleate pool following the feeding of a cholesterol diet supplemented with corn oil. In the present report, an expansion of the monounsaturated, linoleate, and saturated liver ester pools was also noted following the

CHOLESTEROL

ESTER

feeding of cholesterol with and without fatty acids. It was consistently observed that the greatest increase in liver cholesterol esters occurred in the monounsaturated fraction. This specific increase in liver cholesterol oleate was not eliminated by the feeding of highly unsaturated fatty acids such as linoleic and arachidonic acids. Perhaps the defect which leads to the increase iu cholesterol oleate is in the over-all transport mechanism of cholesterol, and even though polyunsaturated fatty acids are available for esterification, the other necessary conponents for lipoprotein synthesis are not available in sufficient amounts to transport and metabolize cholesterol. The increase in liver cholesterol esters was riot due entirely to exogenous cholesterol. It is shown, in agreement with the findings of Klein and I\Iartin (27), that there is a large expansion of the endogenous cholesterol ester pools of the liver when cholesterol is fed. It is doubtful that this increase in andogenous esters was derived from synthesis within the liver since it has been amply denlonstrated that, cholesterol synthesis is depressed in the cholesterol-fed animal. A comparison of the specific activities of the free and ester fractions in the liver with comparable data obtained on lymph (28) suggests that the major source of the excess endogenous cholesterol is the intestinc. In this connection, it was previously shown (29) that an endogenous pool of free cholesterol exists in the intestinal wall with which incoming cholesterol is mixed prior to its ester&cation and transfer to the lymph.

553

METABOLISM

5. SWELL, L., FIELD, H., JR., AND TREAUWEI~L, C. R., J. Biol. Chem. 212, 141 (1955). 6. SWELL, L., BYRON, J. E., AND TREAI)WEI,L, C. I~., J. Biol. Chem. 186, 543 (1950). 7. DEYKIN, I)., AND (;OOL)MAN, D. S., Riochem. Hiophys. ZZes. Conunzcn. 8, 411 (1962). 8. SWELI,, I,., ANI) TREADWELL, C. I<., Proc. Sot. Exptl. Hid. Med. 110, 55 (19G2j. 9. DAII,EY, 11. E., SWELI,, L., ANI) TREA~U’EI~I., C. I<., A-lrch. Biochem. Hiophys. 99,334 (1962). 10. SPERRY, W. M., J. Hiol. (‘hem. 111, 467 (1935). 11. KARMEN, A., WHY’I’E, M., ANI) (:OOL)MAN, D. S., J. Lipid Kes. 4, 312 (1963). 12. Lossow, W. J., BROT, N., ANI) CHAIKOFF, I. I,., J. I,ipicl Kes. 3, 207 (1962). 13. KLEIN, P. D., AND MARTIN, Ii. A., J. Hiol. Chem. 234, 1585 (1959). 14. SWELL, L., LAW, M. D., FIEI,D, H., JR., ANI) TREAI~WELI,, C. I< ., .J. Biol. (‘hem. 236, 1960 15.

(1960). SWEI,I., JR.,

L.,

FIELU,

ANU

18.

19. 20. 21.

22. 23. 2-2.

1~. D., M.,

znvest. 2. SWELL,

3.

4.

ANI)

34, 1369 I,.,

TREAINEI.I.,

Ml

(1958). J.

C. A.,

AND

WILLIAMS,

Acta

58, 398 (1962).

CiOoDMAN,

S.

O.,

J.

(Xin.

li.,

JR., J.

PARKER,

R.

D. S., J. Clin.

Z’roc.

P. E., Sot.

itm.

F. T., NICHOLS, J. (Yin. Nub-.

A. V., AND 9, 13 (1961).

WILLS,

D., ANV GO(II) D. S., J. Riol. 237, 3849 (1962). 27. KIXIN, P. I>., ANV MAR’I’IN, II. A., J. Bid. Chem. 234, 3129 (1959). 28. SWELL, L., TROI:T, E. C., JR., HOPPER, J. Ii ., FIELD, H., JR., AND TREADWELL, C. It., J. Biol. Chem. 232, 1 (1958). 26.

DEI-KIN,

(‘hem.

(1955).

TROTJ’J~, 15. C.,

ANL)

GLOI\ISE:~‘,

BYERS,

SCHOOLS,

Mecl. M., (TRIMMER, U., GLASER, A., ANV VOIO~~, K. D., Biochem. Z. 336, 1 (1962). HOHEIM, P. S., HAFT, D. E., GIDEZ, L. I., WHITE, A., ANV EI)ER, H. A., J. Clin. Invest. 42, 1277 (19G3). SWELI,, L., STIJ’I’ZMAN, E., LAW, M. D., ANV TREAUWELL, C. It., ;lrch. Biochem. Hiophys. 97, 383 (1962). HIRSCH, J., AND AHRENS, E. H., JR., J. Riol. Chem. 233,311 (1958). SPERRY, W. M., ANU WEBB, M., J. Biol. Chena. 187, 97 (1950). SWEU., I,., LAW, M. I)., ANY TREADWELI,, C. I~., .Irch. Biochem. Biophys. (In press). KI~EIN, 1’. D., ANV JANSSEN. E. T., .J. Biol. Chem. 234, 1417 (1959). SWELL, I,., LAW, M. D., AND TREAIIWELL, C. Ii., J. A:&. 81, 263 (1963). SWE~, L., AND TREA~wELL, C. It., And. Biochem. 4, 335 (1962).

25. LINIXZREN, 1. FRIEDMAN,

JR.,

C. Ii., 103, G51 (1960).

Exptl. Rid. 16. APOSTOLAKIS, 17.

H.,

TREADWELL,

H.,

Prsm,

F.,

H.,

Chew.

Hiol.

TJAUEN,

Biochim. hvest.

41,1886

JR.,

230, M.,

Biophys. (19G2).

29.

SWEI,I+ L., TROVT, FIEI~U, H., JR.,

.I. Biol.

C’hem.

E. C., JR., HOPPER, AND TREADWELL,

233, 49 (1958).

J. R., R.,

C.