Studies of the distributions of lipids in hypercholesteremic rats

ARCHIVES

OF

BIOCHEMISTRY

Studies 3. Changes

AND

BIOPHYSICS

of the Distributions

270-283 (1965)

110,

of Lipids

in Hypercholesteremia

in Hypercholesteremic

Rats

and Tissue Fatty Acids Induced

by Dietary

Fats and Marine JAMES

J PEIFER,3 University

Oil Fractions1~2

W. 0. LUNDBERG, of Minnesota,

S. ISHIO,”

The Hormel

Received

October

Institute,

ilND

Austin,

E. WARMANEN Minnesota

1, 1964

The hypocholesteremic activities of dogfish liver oil and tuna and menhaden oils were duplicated by feeding rats proportionate amounts of the fatty acid components of the oils; neither selachyl alcohol nor the unsaponifiable components of the oils affected the hypercholesteremia of the rats. The relative hypocholesteremic activities of menhaden oil fatty acid ester fractions (1.17. of 48.6-353) were not predictable on the basis of such criteria as their total unsaturation, contents of polyunsaturated fatty acids (PUFA), or contents of more saturated acids. The hypocholesteremic activity of the fraction with an iodine value of 48.6 was similar to that of corn oil (I.V. = 124). Only minor amounts of the linolenate homologues were required to promote significant reductions in the cholesterol contents of the livers in these rats. Rats fed tallow-PUFA mixtures incorporated little of the available PUFA into the cholesterol esters of their tissues. However, a homeostatic mechanism appeared to regulate the incorporation of appreciable and uniform amounts of available PUFA into the plasma phospholipids and myocardia of the rats. The PUFA patterns in the plasma and liver suggested that the preconditioned animals had a partial depletion of the essential fatty acid (EFA) reserves for these tissues. However, a very adequate supply of EFA was apparently available for the myocardial tissues of these same rats. The fractionation of marine oils and some modified methods for lipid analyses are discussed.

Supplements of both the linoleate and linolenate families5 of acids have exhibited 1 Research supported by grants from the American Heart Association, the Minnesota Heart Association, U.S. Department of Interior, Fish and Wildlife Service, U.S. Public Health Service grant HE 04386, and The Hormel Foundation. 2 Partially presented at the 17th Annual American Heart Association Council Meeting, Los Angeles, California, October, 1963. 3 These studies were completed during the tenure of an Established Investigatorship from the American Heart Association. 4 Present address : Department of Fisheries Chemistry, Faculty of Agriculture, University of Kyushu, Fukuoka, Japan. 5 A classification of acids according to families having their first double bonds similar distances from the terminal methyl group of the acid (16).

hypocholesteremic activit,ies in man (l-3) and experimental animals (4-9). Previous studies demonstrated that, pure linolenate, tuna and menhaden oils, and cod liver oil were more effective t’han pure linoleate in alleviating the hypercholest’eremia of rats fed cholesterol (5, 6). The marine oils were rich sources of the more highly unsaturated homologues of linolenatej (1, 5, 7), whereas Linoleate and arachidonate, members of the have their first two double “linoleate family,” bonds at the 6 and 9 positions from the terminal methyl group. Linolenate and the eicosapentaenoic and docosahexaenoic acids formed endogenously from linolenate (5, 16) have their first three double bonds at the 3, 6, and 9 positions from the terminal methyl group, and all three acids belong to the “linolenate family.” 270

HYPERCHOLESTEHEMIA,

linoleate represented the major source of polyunsaturated fatty acids (PUI:A) available from conm~on dietary fats. The relative hypocholesteremic activit)ies of the oils appeared to be dependent upon the total unsat8uration available from their I’UFA componenls (<5),However, further investigations revealed that, factors ot’her than the total unsaturation must be part.ially responsible for the effects of the marine oils (7) and higher homologues of bot’h families of 1’UlcA (10). This may have been due to the hypocholesteremic effects of unsaponifiablc components found in some marine oils (II), a possible synergistic action between the different fatty acids in the oils (4), and/or a great)er hypocholest,eremic activity associated with specific types of PUFA (10). Exogenous cholesterol (12) and high caloric int,akes of salurated fat (13) promote an increased acacumulation of the higher homologues of both the linoleate and linolenate families of acids into the myocardium of a rat’. Oils rich in linoleate have also been reported to promote an accumulation of exogenous cholesterol in t’he cardiovascular tissues of the rat (14). Recent studies suggest, t)hat diet’ary cholesterol significantly elevates the circulating lipid levels of man and t,ends to negate much of the hypocholesteremic effects of “linoleak rich oils” in these individuals (15). These observations suggest that more at’tention should be given to t)he metabolic fat’e of different PUFA in cholest,erol-fed rats which receive most of their fat calories from more sat#urat,ed acids. Such information may provide a better understanding of t.he interdependence of the metabolic effect)s of exogenous cholesterol, different I’UFA, and/or sat,urated fats on the lipid patterns of cardiovascular tissues. The present, report describes the effect)s of different marine oil components and various levels of different types of PUFA on the plasma, liver, and myocardial tissues of hypercholesteremic rats. Hypercholesteremia was induced by feeding the rats a diet containing cholesterol and bile Sal&. Beef tallow was a major source of t,he fat calories during both the preconditioning and test periods of the experiments.

FATS,

APr’L, AIARINE

OILS

MIZTERIALS

AND

271 METHODS

Male rats, Sprague-Dawley strain, weighing 250-300 gm each, were made hgpercholesteremic by feeding them a diet containing 0.50/; cholesterol, 0.5’);, ox bile, and either 10 or 15% beef tallow. After a minimum preconditioning period of 5 weeks, the hypercholesteremic rats were distributed into groups having similar mean plasma cholesterol levels and body weights. These groups then received diets in which part of the tallow had been replaced by an equivalent amount of the different test lipids. The compositions of the diets are recorded in Table I. In Experiments I and II, the test lipids were given to the preconditioned rats over a 5.week supplementation period. In the third experiment, the preconditioned rats received the test lipids for 7 weeks. All groups received one third, or more, of their total calories from tallow; cholesterol and ox bile were constant components in both the preconditioning and test diets. Food and water were supplied ad Zibitunz during the course of the experiments. TABLE

I

COMPOSITIONS OF EXPERIMEKTAL

Components Casein, vitamin test” Alpha-cellulosea Wesson salt mixture11 Sucrose Cholesterol B-vitamin mix* Fat -sol. mixc Ox bile powde+ Beef tallowe Test lipid

lS’-GFat

diet

18 4 4 56 0.5 2 0.1 0.5 4.9 10

DIETS lO%$at

18 4 4 61 0.5 2 0.1 0.5 6.6 3.3

u Nutritional Biochemicals Corporation. h This glycose mixture supplied the following vitamins in mg/lOO gm diet: ascorbic acid, 5; Ca pantothenate, 4; inositol, 10; niacin, 4; p-aminobenxoic acid, 10; riboflavin, 0.5; pyridoxine-HCl, 0.2; thiamine-HCl, 0.5; folic acid, 0.2; biotin, 0.05 and 0.005 B12; and choline chloride, 100. c This hydrogenated coconut oil mixture supplied the following fat soluble vitamins in mg/lOO gm diet: vitamin A, 0.67; a-tocopherol, 5; a-tocopherol acetate, 5; calciferol, 0.002; and menadione, 0.02. These diets had 3-6 mg, or more, of a-tocopherol for each gram of PUFA present in the tallow test lipid mixtures. d Fisher Scientific Company. 6 Purified edible beef tallow obtained from The Hormel Company, Austin, Minnesota.

272

PEIFER,

LUNDBERG,

ISHIO,

Selachyl alcohol (see Table III) had been isolated from natural sources; TLC and reversedphase chromatography indicated that it was better than 96% pure selachyl alcohol. The butter and margarine supplements were added to the diets without attempting to remove their nonlipid components (see footnotes of Table II). The marine oils tested were the liver oil of dogfish (Squalus acanthias) and the whole body oils of tuna (Thunnus thynnus) and menhaden (Brevoortia lyrunnus) . The marine oils were saponified at 60°C with two volumes of 5.6 IM KOH-50% methanol. The unsaponifiable materials were removed by repeated extractions with petroleum ether (b.p., 36”-6o”C), and these extracts were then washed

AND

WARMANEN

free of fatty acid soaps. The free fatty acids were recovered in petroleum ether after the combined soap solutions had been acidified with 6 N HCl. Ethyl esters were prepared by stirring a mixture of three volumes of absolute ethanol-3% concentrated sulfuric acid with one volume of fatty acids for 18-24 hours at 60°C. All operations were carried out under a blanket of pure nitrogen. The fatty acids from one sample of menhaden oil were further fractionated by low temperature crystallization (17), urea adduct formation (18), and alembic distillations. The fractionation procedures are summarized in Fig. 1. The distilled esters were sealed under pure nitrogen and stored in a dry ice-deep freeze chamber until needed (5,7). Gas-liquid chromatographic (GLC) analyses

MENHADEN OIL ACIDS LV.= 187 (I.1 KQ acb I IO L petrol. ether)

If

L-l

I

-72O

MO-I I.%=49.6 (20.6%)

Et.Esters 3partsUreo in ethanol I .v.= 93 (21.2%)

I.V. =I27 ( 9.5 %I

Solid Urea Complex

1

‘.I$0 P.Distil. i35.169“ .3mm

MO-4

I.v.= 266 (19.3%)

Crystal.

Filtrate

I

I. H20 2Distil. 169-196°

.3mm

MO-5 l.V.6363 (l6.9%)

FIG. 1. The preparation of the five menhaden oil fractions. The low temperature crystallizations at -10°C and -72°C allowed isolation of the more saturated fractions MO-1 to MO-3. The fraction which remained soluble at -72” (L2) was esterified and further fractionated by forming the urea adducts. Fraction MO-4 was the fraction recovered from the solid adduct, while MO-5 was that obtained from the filtrate; the esters were released from the urea complexes by dilution with water. All five fractions were alembically distilled under the conditions of temperature and pressure indicated in the figure. The iodine values and percentage yields (i.e., the values in parentheses) are recorded for each fraction.

HYPERCHOLESTEREMIA, were performed with an instrument” equipped with a p-ionization detector. Fractionations were done with glass columns, 4 ft X 4 mm, which had been packed with either LAC-2R-446’ (15% on Celite, 160°C and 12 psi argon) or diethylene glycol succinate (DEGS)’ (25Oj, on 80-100 mesh Chromosorb W,’ 180°C and 10 psi argon). The ethyl esters of the marine oils were analyzed with both the LAC and DEGS phases in order to differentiate overlapping components (19). Methyl esters prepared from the liver lipids (see Table VII) were analyzed with DEGS treated with 2% phosphoric acid according to the procedure of Metcalfe (20). We have found that phosphorylated-DEGS columns can sometimes be used for more than 4 months for the efficient fractionation of fatty acid esters at 180”195°C. Identification of the components was based on their retention times relative to that of stearate (x/18:0). These relative retention t,imes (x/18:0) were compared with those of pure esters* and those already reported for menhaden oil fatty acids (19). Quantitation of the data was calculated on the basis of the products (THf) of t.he absolute retention times (T), the peak heights of the GLC curves (H), and an empirical correction factor (f). GLC methods were standardized by analyzing mixtures which contained various proportions of 8 to 15 different pure fatty acid esters.8 Analyses of such mixtures showed the need for small correction factors (f): 1.0 for 16:0, 18:0, 20:5, and 22:6; 0.9 for 14:0, 18:2, 18:3, 20:1, and 20:4; and 1.2 for 18:l and 22:l (see footnotes of Table II for description of terminology). Where reference compounds were not available (i.e., 22:4), the correction factors were estimated on the basis of those obtained for esters having similar retention times and similar degrees of unsaturation. Such procedures are also used by us when using hydrogen flame detectors; in t,his case, the need for correction factors for the different components is less obvious. Other recent modifications of our GLC methods have been described elsewhere (7). The analytical data for the lipid supplements are listed in Table II, and those of the tallow-test lipid mixtures are shown in Table IV. The alkali isomerization data and the iodine values for fraction &IO-l (I.\-. = 48.6) suggest that it contained small amounts of polyunsaturated acids not readily quantitated by the GLC techniques available at the time of these experiments. Routine monitoring of preparative and fractionation procedures was accomplished by the 6 Pye Argon Chromatographic Instrument. 7 Wilkens Instrument and Research, Inc., Walnut Creek, California. 8 Pure esters obtained from The Hormel Institute, University of Minnesota.

FATS,

AND

MARINE

OILS

273

thin-layer chromatographic (TLC)-microtechnique of the author (21). The silica gel-coated microplates, 3% X 4 inches, were also used for the preparation of the fractions of cholesterol esters, triglycerides, and phospholipids of the liver (see Table VII). Scrapings from one to four of the microplates yielded enough of the fractions for transest,erifications and further analyses by GLC. The advantages and limitations of quantitative TLC on microchromatoplates will be published elsewhere (21). Because of our limited GLC facilities during some of these experiments, some lipids were analyzed only by the alkali isomerization method (5). Iodine values were determined by the micromethod of Luddy et al. (22). Two-ml blood samples obtained by heart puncture were collected at intervals of not less than 3 weeks; Experiment III was initiated with eight animals per group, and only a few animals were lost during the course of this or the other experiments. Tissue lipids were extracted according to the procedure of Folch et al. (23)) and cholesterol was analyzed according to the method of Abel1 et al. (24). Because of the large amounts of cholesterol esters of saturated acids in the livers and plasmas (see Tables V, VI, and VIII), hydrolysis of such esters were always checked by TLC. The column chromatography procedures and other analytical methods and precautions taken to minimize oxidative changes in the dietary lipids have been described in our previous reports (5, 7). RESULTS

We had noted previously that tuna (5) and menhaden oils (5, 7) were very effective hypocholesteremic agents for the bile acidcholesterol treated rats. Table III shows the relative cont’ributions of the unsaponifiable (U) and fat,ty acid components (EE) t’o the hypocholesteremic act,ivities of these two oils. Since these oils contained very minor amount’s of the unsaponifiable components (i.e., 1.5 and 0.8 %, respectively), the rats were preconditioned with diets containing the higher levels of fat (i.e., 15 % fat diet of Table I), and two thirds of t,his was replaced by the total lipids of t’he marine oils (U + EE) during the test periods. Ot,her groups received proport’ionate amounts of the unsaponifiable (U) and fatty acid-ethyl ester fractions (see Table III). Only those groups which received supplements containing the fatty acid components of the marine oils showed a decrease in their circulating cholesterol levels at the end of the 1 month test period. The unsaponifiable fractions did

274

PEIFER,

LUNDBERG,

ISHIO, TABLE

GLC Fatty

xb

acida

18

* 8:O *10:0 *12:0 13:o *14:0 Unsatg *15:0 *16:0 *16:1 16:2-4e *17:0 *18:0 *18:1 *18:2 (linoleic) 18:2 *18:3 18:4 *20:0 *20:1 20:3 *20:4 (arachidonic) 2O:uns.a *20:5 22:50 22:5 *22:6 Iodine

values

ANALYSES OF Tallow

Butt.c

AND

WARMANEN

II

ACID COMPONENTS IN TEST LIPIDS

FATTY ivkqd

Corn

oil’

1.2 2.2 3.1 + 11.2

.06 .ll .18 .24 .33 .38 .44 .56 .68

25.5 1.7

1.5 27.7 2.2

.77 1.00 1.18 1.50

.8 24.9 41.1 1.2

1.1 14.2 27.5 2.1

4.8 65.6 9.5

3.4 28.4 49.9

.3

1.5

.8

.8

MO-lf

MO-2

11.9

1.0 13.9

MO-3

MO-4

MO-5

5.6

1.89 2.07 2.42 1.78 2.10 2.93 3.65

14.8 +

13.0 +

.Q 59.0 5.0 + .Q 6.3 4.9

.8 17.5 21.4

2.0 31.0 1.0

1.1

+h

+

5.5 4.4

7.1 50.5 1.6 +

+ +

2.3

13.8

+

4.25 4.60 7.43 8.74 10.50 40.5

44.3

80

124

1.1 4.6 5.6 .8 2.2 3.1 1.8

5.1

+ 1.8 1.2 1.8 3.1 3.2 1.3

+ + 8.1

+ 47.0 1.7 1.7 20.8

1.4

+

5.6

7.7 27.8

+

+

+

7.1 11.0

93

127

48.6

5.1

266

1.0

353

a Fatty acids marked with an asterisk were used as reference compounds in establishing retention times and correction factors for these GLC analyses (see Materials and Methods). b Retention times relative to that of stearate (18:0) when the phosphorylated-DEGS column was and Methods). The first series of numbers designate the carbon used for these analyses (see Materials chain length of the acids, and those following the colon represent the number of double bonds present in the acid, i.e., 18:2 is an acid having 18 carbons and two double bonds. c Butterfat of butter obtained from Ankeny Bros. Distributing Co., Austin, Minnesota. The better contained 15y0 water and 3.5% nonlipid solids. d Fatty acid composition of margarine lipids obtained from Durkee’s Margarine, NuMaid Products Co., Cincinnati, Ohio. The margarine contained 4.9% nonlipid solids and 15% water. 6 Mazola oil. f Pure triglycerides of 18 carbon acids would contain 95% fatty acids; pure ethyl esters of such acids would contain 92y0 fatty acids. g Components tentatively identified on the basis of data reported elsewhere (19). h The + sign indicates that the component was present in amounts which were below the limits of accuracy for our quantitative methods (i.e., less than 0.5% of total acids). not

significantly

influence

the

circulating

lipid levels of the hypercholesteremic rats. Since the unsaponifiable fractions of some fish liver oils have been reported to exhibit

hypocholesteremic

activities

in

chickens

(II), the relative effects of dogfish liver oil components were tested in Experiment II. Because this oil contained appreciably more

HYPERCHOLESTEREMIA,

FATS, TABLE

EFFECTS OF MARINE

Group

Experiment Ie Tuna oil U + EE FA-esters (EE) IJnsaponif. (U) Menhaden oil IT + EE FA -esters (EE) Unsaponif. (U) Experiment II Dog&h liver oil Whole oil (DFO) FA-esters (EE) Unsaponif. (U) Selachyl alcohol’ Tallow controls

ivo. rats

AND

MARISE

273

OILS

III

OIL COMPONENTS ON PLASMA CHOLESTEROL LEVELS OF RATS” Diet Lipids (gm/lOO gm diet) T?lllOW

Total plasma cholesterolb (mg/lOO ml)

Suppl.

Terminal rng/lOO ml

Initial

p-valuesC

Terminal body es. (gm)b.”

5 5 4

5 5 15

10 10 0.15

327 i 311 f 378 f

32 54 81

146 i 18 154 f 38 481 + 13

.Ol .05

401 i 398 f

4 4 4

5 5 15

10 10 0.08

428 f 316 f 285 f

82 57 40

198 f 164 f 388 f

7 22 112

.03 .05

398 zk 15 (8) 374 f 17 (11)

5 6 G 5 6

6.G 6.6 9.7 9.0 10.0

490 565 579 474 587

71 31 83 60 04

335 314 535 524 712

70 38 86 64 109

.05 <<.Ol

3.4 3.4 0.34 1.0 -

f f f f zt

f f f f f

506 503 531 480 521

10 (9) 20 (14)

+ 8 z!z 11 * 9 zt 15 SC 9

(8) (16) (39) (32) (35)

u The test lipid tallow mixtures were fed to the rats for 1 month in both Experiments I and II. b Mean values & standard error of the means. c p-Values as determined by Students method for small samples; probability values are recorded for only those groups where the probability of a significant difference was greater than 957;. d The values in parentheses are the average body weight gains for the rats during the test periods. 8 In Experiment I, the rats received presaponified oils which had their fatty acids converted to ethyl esters (EE). Other groups received proportionate amounts of the esters (EE) and unsaponifiable components (U) which had been isolated from the presaponified oils (U + EE). f This glyceryl ether was obtained from L. Light & Co., Ltd., Bucks, England. We wish to thank Dr. Helmut K. Mangold of our Institute for giving us this glyceryl ether and his analytical data regarding its purity.

of the unsaponifiable material (i.e., 10% of the whole oil), the rat)s were preconditioned with t,he diet containing 10% fat, and the dogfish liver oil (DFO) replaced only one third of the tallow in the test diets. Two OIher groups were fed proportionate amounts of the unsaponifiable (U) and fatty acid components (EE) present in t,he liver oil (see Table III). Only the whole liver oil and its fat#ty acid components lowered the plasma cholesterol levels of the rats. Since dogfish liver oil and other shark liver oils are especially rich sources of glyceryl ethers (25), the comparat,ive effects of this class of lipids were also invest,igated. However, the hypercholesteremia of the rats was unaffected by their ingestion of a diet in which selachyl alcohol represented 10% of the total lipids (see Table III). The possible effect,s of higher levels of the unsaponifiable

component,s of marine oils were not, investigated. A further study was init,iated in an attempt to correlate the fatty acid compositions of five menhaden oil ethyl est’er fractions (see Tables II and IV) with their respective effect.s on the plasma and tissue lipids of hypercholest’eremic rats. These fract>ions, MO-1 to MO-5, differed in their total unsaturation and their contents of sat’urated, monoenoic, and “linolenate-type” acids (i.e., eicosapentaenoic and docosahexaenoic acids). The coruparatjive effect,s of some coniman dietary fats having different tot)al unsaturation and containing different levels of linoleate were also tested. Table IV shows the compositions of the tallow test lipid mixtures as analyzed by both the GLC and alkali isomerization methods. In t.his experiment,, the test lipids were given as substitutes

276

PEIFER,

LUNDBERG,

ISHIO, TABLE

&O-15:0 16:0 18:O 16:l 18:l 16:2-4 18:2 L 18:3 2O:l 20:3 18:4 + 20:2 20:4 A 20 : unsat . 20:5 22~5s 22:6 Total PUFAb Dienes Trienes Tetraenes Pentaenes Hexaenes %s+oc % PUFA Total FAd

WARMANEN

LIPID

MIXTURES

IV

FATTY ACID COMPOSITIONS OF TALLOW-TEST Fatty acid

AND

Tallow

Butter

Margar.

Corn oil

25.5 24.9 1.7 41.1

5.4 24.9 20.6 1.8 35.9

21.2 18.0 1.1 46.0

21.3 17.7 1.1 36.9

1.2 0.3

1.4 0.6

3.5 0.5

17.4 0.5

(GM/KG

MO1

MO-2

MO-3

4.1 36.7 18.7 2.8 29.0 + 0.8 0.2

5.2 22.8 17.3 8.5 37.7

18.8 19.0 2.2 44.2

0.5

1.1 0.3

+

+

1.0 .2

1.8 .7

3.4 .l

15.5 .4

0.8 .2 .9 1.2 .4

98.5 1.5 94.7

97.8 2.2 90.6

95.6 4.4 90.3

81.1 18.9 94.9

98 2 92.8

MO-4

MO-5

1.9 17.4 17.3 2.3 28.4 1.9 1.4 0.8

17.0 16.6 1.1 27.4 1.7 2.5 0.2

+

1.1 0.4 0.4 2.6 9.3 2.4 3.7

2.7 0.3 + 15.7 1.1 6.9

0.8 .4 1.1 2.6 1.8

2.1 .5 4.5 9.5 5.6

0.7 .l 6.2 16.2 9.7

95 5 92.2

73.1 26.9 91.3

66.7 33.3 93.2

1.3 0.2 4.6

1.9 ?

98.3 1.7 92.9

DIET)”

Q Calculations based on the GLC data for the different supplements (see Table II) and the 6.6 and 3.3 gm of tallow and test lipids, respectively, included in the diets. The diets supplemented with butter and margarine contained slightly less than 107, fat because of the moisture and nonlipid components in these test lipids. b The levels of polyunsaturated acids in the tallow-test lipid mixtures as determined by the alkali isomerization methods. c Per cent “S + 0” is the gm of saturated (S) and monounsaturated (0) acids present in 100 gm of the dietary lipids. Similarly, ‘$&PUFA is the amount of total polyunsaturated acids found in the same amount of the tallow-test lipid mixtures. These values are based on the results obtained by GLC. d Total fatty acids found in the different diets. All calculations were based on a 95y0 fatty acid composition for triglycerides and 92% fatty acid composition for the ethyl esters.

for one third of the tallow present in the 10 % fat preconditioning diet. Figure 2 illustrates the progressive changes in the hypercholesteremia of the rats following ingestion of the test lipids. The low levels of PUFA in the diets of the tallow controls and butter-supplemented groups (i.e., 120 and 140 mg linoleate in each 100 gm diet, respectively) allowed the rats to develop a slightly greater hypercholesteremia during the 7-week test period. When rats received 4.4 % of t’heir fat, calories from t’he tot’al

PUFA of the margarine-supplemented diet, the severity of their hypercholesteremia remained unchanged. However, ingestion of fats containing 18.9-33.3 % PUFA (corn oil and the MO-4 and MO-5 supplements) promoted significant decreases in the circulating cholesterol levels by the fourth week. By the seventh week, even the most saturated menhaden oil fraction, MO-l (PUFA $ 3.5 %), had lowered the plasma cholesterol levels to that of rats which had received supplements of corn oil. The supplements of corn oil and

HYPERCHOLESTEREMIA,

FATS,

AND

44 ,4------,

I

I

I

I

2 WEEKS

ON

MARINE

277

OILS

-----ma--TALLOW

I

I

4

I

6

I

I 6

SUPPLEMENTS

FIG. 2. The progressive changes in the hypercholesteremic condition of the rats fed common dietary fats and three of the five ester fractions of menhaden oil. Each point represents the mean cholesterol value obtained from the number of rats shown in Table V.

the five menhaden oil fractions also promoted significant reductions in the levels of phospholipids, free and esterified cholesterol, and the cholesterol-phospholipid ratios (TC/ TP) found in the plasmas (Table V). However, the hypocholesteremic effects of the menhaden oil fractions, when compared with those of corn oil and margarine, were much greater than could have been predicted on the basis of their total unsaturation or their contents of PUFA. Although MO-l (I.V. = 48.6) and corn oil (I.V. = 124) were significantly different, in their total unsaturation and patterns of PUFA, both supplements appeared to have similar hypocholesteremic activities by the end of the seventh week of the test period. Fraction MO-5 (I.V. = 353) was also less effective than M-4 (I.V. = 262) in lowering the plasma lipids, although the latter supplement contained significantly less PUFA. Furthermore, the observed effects were not readily correlated with the relative contents of saturated and monosaturated acids (S + 0) found in the diets

(see “c” of Table IV). Diets cont)aining butter, MO-l, and MO-2 had similar levels of “S + 0” and total PUFA, but these same supplements had significantly different effects on the plasma lipid levels of the rats. Despite the differences found in the plasma lipid levels of rats fed t,allow, t,allow-margarine, and tallow-MO-3, all t’hree groups received similar proportions of palmitate, oleate, and stearat’e in t’heir dietary fat,s (Table IV). More lipids tended to accumulate in the livers of rats fed margarine and butter than in the tallow controls. However, ingestion of the menhaden oil fractions promoted the removal of sterols from this tissue (Table VI). The group fed MO-4 had 29.5 % less total lipids in their livers than that found in the tallow controls. Cholest’erol esters were t’he major components in the liver lipids of these cholesterol + bile acid-fed animals. Although corn oil had been effective in lowering the cholesterol levels of the plasma, only the menhaden oil fractions promoted signifi-

278

PEIFER,

LUNDBERG,

ISHIO,

cant reductions in the sterol ester contents of t,he livers. In each experiment, the different groups had similar rates of growth (see values in t)he

Group

No.

IN CIRCULATING

V

LIPIDS

INDUCED

Plasma lipits

(mg/lOO

CEb

FCb

763 735 590 437 358 292 206 165 231

58 50 39 24 32 26 16 11 21

rats TC

524 499 401 297 255 206 142 112 16Q

Tal-control Butter Margarine Corn oil MO-1 MO-2 MO-3 MO-4 MO-5

f f f It f b f f f

52 44 27 19 36 19 14 11 32

WARMANEN

parent’heses of Tables II and VI), and t,he rats showed no apparent, aversion t,o the diets containing t,he tallow-test lipid mixtures.

TABLE CHANGES

AND

BY SUPPLEMENTS ml plasma)’

TC/PL

PL

145 151 142 123 126 113 125 77 89

* f f zk 3~ f f * h

FA’CE+PL

7 11 9 7 5 11 5 8 5

436 428 360 279 247 209 180 128 165

3.6 3.3 2.8 2.4 2.0 1.9 1.2 1.5 1.8

a Plasma lipid levels after 7 weeks of treatment with the different supplements. Mean values i the standard errors of the means are recorded for the total cholesterol (TC) and total phospholipid (PL) levels of the plasmas. b The concentrations of cholesterol esters (CE) and free cholesterol (FC) were obtained by analyses of pooled samples fractionated on silicic acid columns (4). c FA~E+PL are the total fatty acids which had circulated as components of cholesterol esters and phospholipids. This represents the major portion of the plasma fatty acids since triglycerides were relatively minor components in these rats which had been fasted overnight prior to the collection of blood samples.

TABLE EFFECTS Liver Group

Tallow Butter Margarine Corn oil MO-1 MO-2 MO-3 MO-4 MO-5

weights kw/100

OF SUPPLEMENTS

Total liver lipids’

b.pwl;b

(pm)

(%A)

Lipids/l00 gm liver (pm)

5.4 5.8 5.7 5.5 5.5 5.5 5.5 5.0 5.0

4.7 5.4 5.2 4.7 4.1 3.5 3.5 3.3 3.8

+14.9 +10.6 -12.8 -25.5 -25.5 -29.5 -19.0

23.7 24.8 25.2 21.8 19.8 18.5 17.3 17.7 20.4

Lipids/whole

liver

VI

ON LIVER cEd/loo gm liver km)

16.9 18.9 18.4 16.3 12.6 12.5 12.3 13.8

LIPIDS

AND

GROWTHO

Camp. liver lipids (gm/lOO gm lipid) CE

FC

TG

PL

71.2 76.2 73.0 75.5 63.6 72.3 69.6 67.8

.6 .6 .5 .6 .7

24.4 20.2 23.8 21.1 31.8

.9 .9 .9

19.6 24.4 27.4

3.8 3.0 2.8 3.6 3.9 7.0 5.1 3.9

Terminal b.w. (gm)”

363 373 363 387 376 340 364 383 375

f 14 (58) f 9 (66) f 10 (58) f 18 (70) f 12 (67) z+z 16 (48) + 9 (84) f 19 (48) zk 19 (68)

a Data obtained by analyses of pooled liver lipids from the numbers of rats shown in Table V. b The mean liver weights for all groups were statistically equivalent. b.w. = body weights. c The average total lipid content in the whole livers of each rat. The percentage change ($$A) in liver lipids refers to the differences between total liver lipids of the groups fed the test lipids and their tallow controls. d Results from analyses of fractions obtained by silicic acid column chromatography (4). CE refers to cholesterol esters, FC to free cholesterol, TG to triglycerides, and PL to phospholipids. e Terminal body weights f standard error of the means. The values in parentheses are the mean gains in body weight during the 7-week supplementation period.

HYPEIZCHOLESTEl~F,hlIA,

FATS,

Tissue fatty acid pattems. Comparatively lit tde of the stearate or polyunsaturated acids made available in the diet,s were iucorporated iut,o the sterol esters of the livers (coinpare dat,a of Tables IV and VII-A). Significant quantities of PUI’A were fouud ouly in the cholesterol esters of rats which had received supplements of corn oil arid fract,ion :\IO-4. However, t,he 6.5 and 16.8 % levels of PUFA it1 the sterol esters of these two groups were far below the contents of PUFA in their iugested lipids. The disproportionately large aulouuts of monoeuoic acids esterified

Group

16:0

16: 1

OF FATTY 18:O

18: 1

ACIDS

LIPID

18:3

Moles acid/i00 23 21 19 18 21 20 20 16 20

15 16 15 18 18 20 15 21 16

3 3 3 3 4 4 3 3 3

59 59 62 55 53 53 61 44 59

Tall. Butt. Mary. C.O. 110-l MO-2 120-3 RlO-4 RlO-5

105 105 102 102 96 84 66 42 60

21 21 18 18 15 21 12 6 12

27 30 33 30 24 33 36 45 30

120 117 126 101 101 104 104 104 87

Tall. Butt. Marg. C.O. 1\1(.)-1 n10-2 MO-3 MO-4 MO-5

36 36 32 32 50 48 50 40 44

10 18 G 14 12 8 8 8 8

48 28 58 28 40 40 40 32 40

.4 .4 .4 4.3 1.1 .9 .4 2.1 .4

ESTERS 20:3

20:4

OF THE 20:u

LIVERS” 20:s

“?$+

22~6

95 PUFA

2 +

1.2 1.2 1.2 6.5 3.9 3.1 2.2 16.8 3.2

moles ester

A. Cholesterol Tall. Butt. Marg. C.O. MO-1 MO-2 MO-3 MO-4 MO-5

279

OILS

VII IN

1x:2

MARINE

with cholesterol resulted in cholesteryl oleate beiug a major lipid cornpouent~ in the livers of all groups. The triglycerides of the liver (Table VII-B) contained considerably more PUFA than the sterol esters, and the proportions of oleic and pahuit’ic acids in t’hese neutral esters were more similar to t,hose of the ingested lipids. The higher levels of I’UFA supplied by corn oil, 1110-4, and MO-5, promoted proportionately greater accuruulations of polyunsaturated acids into these triglycerides. Even t,he trace amounts of

TABLE DISTRIBUTIONS

AND

esters

.8 .8 .8 1.2 .8 1.2 .8 .8 .8

1

2

2 1 1 7 2

2

l?. Triglycerides 3 3 5 15 3 4 4 7 4 C. Total 5G GG 56 44 58 50 4G 58 40

9 14 10 42 8 7 7 G 4

6 6 6 + 3 3 5 2 2

9 12 9 18 6 9 9 12 9

3 3

3 3 + 3 3

3 6 3

3 9 9 12 24 27

3 3 3 9 12 21

2 2 2 4 4 4 8 4 16

4 4 4 4 4 6 10 18 10

15 15 15 33 45

8 10 8 13 14 15 17 31 37

phospholipids 2 2 2

18 14 16

2

4 2 2 2

2

10 10 10 30 G 10 10 6 12

4 14 4 8

a GLC-analyses were carried out on fractions obtained by preparative on microchromatoplates (see Materials and Methods) (21).

thin-layer

4 4 2 8 10 14 1-l 24

24 25 23 40 20 27 28 29 33

chromatograph

280

PEIFER,

LUNDBERG,

ISHIO,

PUFA available from MO-l to MO-3 caused significant increases in the 20: 5 and 22: 6 levels of these esters. The phospholipids were the richest sources of PU’FA in the livers of seven of the nine groups (Table VII-C). Rats which had received 17.4 % of their fat calories as linoleate (i.e., from the corn oil-tallow mixture) distributed approximately twice as much linoleate and archidonate into each mole of phospholipid than into the neutral glycerides of the liver. However, similar quantities of total PUFA were found in both the triglycerides and phospholipids of rats which had received 26.9 and 33.3 % PUFA from the supplements of MO-4 and 310-5. In the phospholipids, stearic acid was a relatively predominate component, whereas the molecular concentrations of oleic acid were one third to one half of those on the triglycerides. There was also an apparent preferential retention of some exogenous PUFA in the triglycerides and phospholipids. Both contained more 22: 6 than 20: 5 even when the rats received twice as much eicosapentaenoic acid in their diets (i.e., groups MO-4 and MO-5). Only small amounts of A588, “-eicosatrienoic acid (20:3) were found in the triglycerides of the liver; the essential fatty acid index (20:3/20:4) (10) was only 0.7 in the triglycerides of the tallow controls. Because TABLE DISTRIBUTION

OF FATTY

AND

WARMANEN

of an apparent preferential distribution of the trienoic acid into the phospholipids, the ratio of 20: 3/20: 4 in the phospholipids of these same animals was 1.8. This suggests that the preconditioned rats may have been near the borderline of an EFA deficiency (10). However, corn oil and all five fractions of menhaden oil eliminated this sybdrome of essential fatty acid deficiency. The PUFA of the circulating phospholipids also reflected the qualitative differences of the ingested lipids (Table VIII). Linoleic and arachidonic acids (i.e., the dienoic and tetraenoic acids, respectively) were elevated in the phospholipids of those rats fed the linoleate-rich corn oil supplement. Similarly, the menhaden oil fractions promoted the accumulations of pentaenoic and hexaenoic acids into these phospholipids. However, the distributions of the polyunsaturated acids were much more uniform than could have been predicted on the basis of the compositions of the diets. One to seventeen per cent linoleate was present in the tallow control and corn oil-supplemented diets, whereas the marine oils supplied from trace amounts to 22% of the eicosapentaenoic and docosahexaenoic acid mixtures to the diets (i.e., MO-l and MO-5, respectively). Kevertheless, all groups incorporated relatively similar amounts of total PUFA into their plasma phospholipids (i.e., 36 and 31% of t’otal VIII ACIDS

IN PLASMA

LIPIDS’ Cholesterol esters (moles/100 moles ester)

Plasma phospholipids (moles/100 moles ester) Group

Tallow Butter Margarine Corn oil MO-l MO-3 MO-4 MO-5

Diene

Triene

Tetraene

Pentaene

Hexaene

Total PUFA

22 32 37 57 10 28 17 13

17 9 17 6 4 3 0 0

15 10 16 31 8 19 11 12

5 3 5 8 9 16 9 17

14 13 12 16 16 24 32 22

72 66 87 117 47 90 69 63

s+o

128 134 113 83 153 110 131 137

PUFA

3 2 3 8 2 7 7 7

s+o

97 98 97 92 98 93 93 93

a Pooled plasma lipids were fractionated on silicic acid columns. The cholesterol ester and phospholipid fractions were further analyzed for their polyunsaturated acid components by the alkali isomerization (AI) method. The saturated and monounsaturated components (S+O) were estimated as the differences between the molar content of total acids in the esters and the total PUFA obtained by the AI method.

HYPERCHOLESTEREMIA,

FATS, AND MARINE TABLE

281

OILS

IX

Polyunsaturated fatty acids (mg/lOOgm ventricle)” Group

Tallow Butter Margarine Corn oil MO-1 MO-2 110-3 MO-4 MO-5

Diene

Triene

Tetraene

Pentaene

HCWSIe

Total PUFA

190 172 316 378 132 123 85 83 37

76 G7 55 0 0 0 0 0 0

370 366 417 478 258 262 223 226 196

59 66 52 77 138 145 192 168 174

144 207 137 118 331 355 468 387 530

835 878 977 1051 859 885 968 864 937

GLC-analyses of Heart Lipids (mg/iOOgm ventrick)* 18:2

Tallow MO-4

261 270

20:3

38 29

20:4

20:s

22:s

2216

Total PUFA

16:0

18:O

18:1

1060 560

26 122

58 104

296 543

1739 1628

354 410

339 365

374 392

(1The pooled myocardial lipids from the nine groups were analyzed by the alkali isomerization method. * This GLC data was obtained for heart lipids from hypercholesteremic rats used in a more recent studv. The tallow controls received the same lo%--fat diet listed in Table I, and the MO-4 fraction was fed at the 1% level in the diet. PUFA in t’he plasma phospholipids of the controls and group fed MO-5, respectively),

and groups fed the menhaden oil fractions had comparatively similar patterns of pentaenes and hexaenes in these esters. The sterol esters of the plasma, like those of the liver, contained only minor amounts of total PUFA. The ratio of triene to tetraene acids in the phospholipids of the plasma was 1.1 for the control group. The myocardial lipids reflected differences in the chemical compositions of t’he ingested lipids comparable with those already described for the phospholipids of t)he plasma and liver of these animals (Table IX). Here again, the total PUFA in the hearts of all groups was comparatively similar (i.e., 0.841.05 gm PUFA/lOO gm heart) despite the wide differences in the amounts and patt’erns of PUFA ingested by the nine groups. Both the alkali isomerization and GLC data demonstrated that docosahexaenoic acid was appreciably more abundant than eicosapentaenoic acid in the myocardium of rats fed the marine oil supplements. The relatively minor amounts of trienoic acid in the hearts

would suggest that t’he preconditioned rats had a very adequate supply of essential fatty acids (EFA) in their tissue reserves (12, 13). DISCUSSION

The abundance of sterols in tuna and menhaden oils (7), and the unsaponifiable components of cod liver oil (26), selachyl alcohol, and the other glyceryl ethers of shark liver oils (25) appear to have little influence on the hypercholesteremia of rats. Nevertheless, the studies of Wood and Topliff (11) suggest that the vitamin A contents of some fish liver oils, including dogfish liver oil, might serve as an effective hypocholesteremic agent for cholest’erol-fed chickens. The hypercholesteremia of rats fed bile acids and cholest’erol appears to be a reflection of the double feedback mechanism described by Bheer et al. (27). Such animals accumulate

excessive

amount’s

of cholesteryl

oleate and more saturated esters of cholesterol in their liver and plasma (see Tables VI -VIII). Previous reports (28) suggest that an adequate supply of PUFA would be necessary to mobilize such cholest’erol est’ers from

282

PEIFER,

LUNDBERG,

ISHIO,

the livers of the rat. These reports (see Table VI) and our previous studies (7) indicate that the linolenate homologues from marine oils can effectively mobilize cholesterol out of the livers of hypercholesteremic rats which have been continued on a bile acid-cholesterol regimen. Although we did not attempt to do lipid balance studies in our animals, Kaneda and Alfin-Slater (29) have also found evidence that some marine oils interfere with the absorption of cholesterol by the rat. Although supplements of cholesterol (12) and saturated fat (13) promote an increased accumulation of all types of PUFA in the heart of a rat, such animals usually have less than 0.3 mg of hexaenoic acid in each gram of their myocardium when linoleic acid is their only source of exogenous PUFA. However, hypercholesteremic rats which had received only linoleate (i.e., the tallow controls, linoleate, and butter- and corn oilsupplemented groups) had 0.7-3.0 mg of the six double bonded acid in each gram of their ventricles (see Ref. 5 and Table IX). Further studies will be necessary to establish whether this is a response specifically associated with the stresses of hypercholesteremia in the rat. In our experiments, the exogenous PUFA was distributed from metabolic pools which had been flooded by the more saturated acids of tallow and some of the t’est lipids. Under these conditions, very little of the available PUFA was incorporated into the cholesterol esters of the liver or plasma. However, even trace amounts of the linolenate homologues from menhaden oil were sufficient to cause marked changes in t’he patterns of acids found in the tissue phospholipids and myocardial lipids. Furthermore, a homeostatic mechanism appeared to regulate the distributions of PUFA so that uniform amount’s of available PUFA tended to be incorporated into the myocardium and plasma phospholipids of animals having widely different degrees of hypercholest’eremia and hyperphospholipidemia (see Table V). Furthermore, all groups tended to have similar levels of total PUFA in t’heir hearts whether they received their major sources of PUFA from the linoleate or linolenate family of acids (i.e., from the common dietary fats or the marine oil fractions). This latter effect is

AND

WARMANEN

possibly a reflection of the in vivo competition between different PUFA described by Mohrhauer and Holman (30). The relatively high amounts of docosahexaenoic acids, rather than eicosapentaenoic acid, found in the marine oil-supplemented rats may have been due to a preferential accumulat,ion of the hexaenoic acid in the tissues and/or the in vivo conversion of 20: 5 to 22: 6 (31). The ratios of tirenoic to tetraenoic may be as high as 0.6 in the myocardial lipids of normocholesteremic rats fed linoleate (12, 13). Although eicosatrienoic acid appears to be preferentially incorporated into the t,issue phospholipids (Table VII), the levels of the triene in the tissue phospholipids suggested that t’he EFA reserves available for the plasma and liver had been partially depleted in the preconditioned rats. Nevertheless, these same animals appeared to have a very adequate supply of EFA available for their heart tissue as indicated by the low value of 0.2 for the triene to tet,raene ratio found in the ventricular lipids of the tallow controls (Table IX). Smith (32) has also report,ed that a localized depletion of EFA may occur during pathological changes in the aortic tissues of man. The hypocholesteremic activities of the marine oil fractions (Table V) and whole marine oils (7) do not appear to be predictable on the basis of such criteria as t’otal total polyunsaturat’ion, or unsaturation, their relative contents of saturat,ed and monounsat’urated acids. Factors other than the total available docosahexaenoic and eicosapentaenoic acids must also be partially responsible for the effects of the marine oils and their fractions; fraction MO-5 was the richest source of t’hese two PUFA and yet it was not more effective than the more saturated supplements, MO-3 and 110-4, in lowering the plasma and liver cholesterol levels of the rats. Some type of synergistic action may have been partially responsible for the effectiveness of the more complex mixtures found in MO-3 and MO-4. ACKNOWLEDGMENTS The and D. aspects Mr. M.

authors wish to thank P. Ahn, F. Janssen, Jarvis for their skilled assistance in various of these researches. We also wish to thank Stansby of the Fish and Wildlife Service,

HYPERCHOLESTEREMIA, Seattle, Washington, for the generous supplies the whole marine oils used in these experiments.

FATS, of

REFERENCES 1.

2.

3.

4. 5.

6. 7.

8. 9.

10. 11. 12. 13. l-1. 15. 16.

ENSELME, J., AND CRAWFORD, R., in “Unsaturated Fatty Acids in Atherosclerosis,” pp. 19-30. Pergamon Press, New York (1962). KINGSBURY, K. J., MORGAN, D. M., AYLOTT, C. A., AND EMMERSON, R. Land 1, 739 (1961). IMACHI, K., MICHAELS, G. I)., GUNNING, B., GRASSO, S., FUKAYAMA, G., AND KINSELL, L. W. Am. J. Clin. Nutr. 13, 158 (1963). HEGSTED, 1). M., GOTSIS, A., AND STARE, F. J. J. Nutr. 63,377 (1957). PEIFER, J. J., JANSSEN, F., AHN, P., Cox, W., AND LUNDBERG, W. 0. Arch. Biochem. Biophys. 86, 302 (1960). HAUGE, J. G., AND NICOLAYSEN, It. Acta Physiol. Stand. 46, 26 (1959). PEIFER, J. J., JANSSEN, F., MUESING, R., AND LUNDBERG, W. 0. J. Am. Oil Chem. Sot. 39, 292 (1962). KUHN, S. G., VANDEPUTTE, J., WIND, S., AND YACOWITZ, Y. J. Xutr. 80, 403 (1963). MORIN, R. J., BERNICK, S., MEAD, J. F., AND ALFIN-SLATER, R. B. J. Lipid Res. 3, 432 (1962). PEIFER, J. J. CirclLZation 26, 666 (1962). WOOD, J. D., AND TOPLIFF, J. J. Fish. Res. Bd. Canada 18, 377 (1961). HOLNAN, R. T., AND PEIFER, J. J. J. Nutr. 70, 411 (1960). PEIFER, J. J., AND HOLMAN, R. T. J. Nutr. 68, 155 (1959). GERSON, T., SHORLAND, F. B., AND ADAMS, Y. Biochem. J. 81, 584 (1961). CONNOR, W. E., STONE, D. B., AND HODGES, R. E. J. Clin. Invest. 43, 1691 (1964). MEAD, J. F., in “Lipid Metabolism” (K. Bloch, ed.), pp. 41-68. Wiley, New York (1960).

AND

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283

17. BROWN, J. B., AND KOLBE, R., in “Progress in the Chemistry of Fats and Other Lipids” (Holman, Lundberg, and Malkin, eds.), Vol. 3, p. 57. Pergamon Press, New York (1955). 18. SCHLENK, H. S., in “Progress in the Chemistry of Fats and Other Lipids” (Holman, Lundberg, and Malkin, eds.), Vol. 2, p. 243. Pergamon Press, New York (1954). 19. FARQUHAR, J. W., INSULL, W., ROSEN, P., STOFFEL, W., AND AHREXS, E. H., Nutritional Rev. 17, No. 18, Part II, August (1959). 20. METCALFE, L. D. Nature 188, 142 (1960). 21. PEIFER, J. J. Mikrochim. dcta 3, 142 (1962); other manuscripts in preparation. 22. LUDDY, F. E., BARFORD, I<. A., RIEMENSCHNEIDER, R. W., AND Evass, J. D. J. Biol. Chem. 232, 843 (1958). 23. FOLCH, J., LEES, ill., AND STASLEY, G. H. J. Biol. Chem. 196, 357 (1952). 24. ABELL, L. L., LEVY, B. B., BRODIE, B. B., AND KENDALL, F. E. J. Biol. Chem. 226, 497 (1957). 25. HILDITCH, T. P., in “The Chemical Composition of Natural Fats.” Wiley, New York (1941). 26. DEGROOT, 8. P., AND REED, S. A. Xature 183, 119 (1959). 27. BEHER, W. T., BAKER, G. D., AND AXTHONY, W. L. Proc. Sot. Exptl. Biol. Med. 109, 863 (1962). 28. ALFIN-SLATER, R. B., AFTERGOOD, L., WELLS, H. F., AND DEUEL, H. J. Arch. Biochem. Biophys. 62, 180 (1954). 29. KANEDA, E., AND ALFIN-SLATER, R. B. J. Am. Oil Chem. Sot. 40, 336 (1963). 30. MOHRHAUER, H., AND HOLMAN, R. T. J. Nub. 81, 67 (1963). 31. KLENK, E., AND MOHRHAUEH, H. Hoppe Seyler’s 2. Physiol. Chem. 320, 218 (1960). 32. SMITH, E. Biochem. J. 88,49 (1963).