Interrelationship of stearic acid content and triacylglycerol composition of lard, beef tallow and cocoa butter in rats

539

Interrelationship of Stearic Acid Content and Triacylglycerol Composition of Lard, Beef Tallow and Cocoa Butter in Rats Cathy C. Monsma and Denise M. Ney* Department of Nutritional Sciences, University of Wisconsin, Madison, Wisconsin 53706

We investigated modes whereby stearic acid (18:0) exerts a neutral or cholesterol-lowerlng effect using dietary fats which provided graded levels of 18:0 and distinct triacylglycerol (TAG) profiles. Male Sprague-Dawley rats (150-175 g) were fed diets containing 0.2% cholesterol and 16% fat from corn oil, or from 1% corn oil plus 15% lard (13.2% 18:0), beef tallow (19.2% 18:0) or cocoa butter (34.7% 18:0) for 3 wk, and then killed in a fasted or fed state. Chylc~ micron (CM) fatty acid profiles suggested reduced absorption of 18:0 with greater 18:0 intake. CM TAG profiles in- dicated a reduction or loss of two TAG species compared to the TAG profiles of the stearate-rich diets: 1-palmltoyl- 2~leoyl~stearoyl glycerol (POS) and 1,3<iisteamyl-2~oleoyl glycerol (SOS). Hepatic total cholesterol concentrations were 54-77% lower {P < 0.01) in the cocoa butter-fed than the lard- and beef tallow-fed groups. The cocoa butter group showed a significantly lower ratio of high<lensity lipoprotein esterified/fres cholesterol than all other groups. Hepatic stearoyl-CoA and oleoyl-CoA concentrations, the substrate and product for hepatic A9 desaturase, were not significantly different for corn oil-fed and cocoa butte~fed groups in spite of a large difference in 18:0 intake. These data suggest that the neutral or cholesterol-lowering effect of 18:0 is not due to hepatic conversion of stearic to oleic acid, and that POS and SOS are poorly absorbed from stearate~rich dietary fats. Lipids 28, 539-547 (1993}.

Studies in humans (I-5) and experimental animals (6,7) suggest that ingestion of stearic acid (18:0) has a neutral or hypocholesterolemic effect in contrast to lauric {12:0), my- ristic {14:0) and palmitic {16:0) acids. Thus, controversy has arisen regarding the classification of stearic acid as a "saturated fat" for dietary recommendations and nutritional labeling purposes (8). This controversy reflects a lack of understanding of the mechanisms responsible for the neutral or hypocholesterolemic effects associated with ingestion of stearic acicL Proposed mechanisms include poor absorption of stearic acid from dietary fat (9-12), rapid conversion of stearic acid to oleic acid by hepatic A9 desaturase {13) and effects of the stereospecific triacylglycerol {TAG) structure of stearate-rich dietary fats on their absorption and metal> olism {14-16).

Although many studies have examined the effects of feeding stearate-rich fats {commonly defined as having greater than 12% of fatty acids as 18:0), differences in experimental design make it difficult to draw conclusions about

*To whom correspondence should be addressed at Department of Nutritional Sciences, 1415 Linden Drive, Madison, WI 53706.

Abbreviations: ANOVA, analysis of variance; C n, total acyl carbon number; CM, chylomicron; E/F, esterified/free; EC, esterified cholesterol; FAME, fatty acid methyl esters; GC-FID, gas chromatography-flame-ionization detection; GLM, general linear models; HDL, high-density lipoprotein; LDL, low~lensity lipoprotein; L, linoleic acid; O, oleic acid; P, palmitic acid; POP, 1,3-dipalmitoyl-2-oleoyl glycerol; POS, 1-palmitoyl-2-oleoyl-3-stearoyl glycerol; SOS, 1,3-distearoyl-2- oleoyl glycerol; TAG, triacylglycerol; TG, triglycerides; VLDL, very low density lipoprotein.

the mode{s) of action of stearic acid (17). Using only the fasted state, often a single stearate-rich fat was compared to either a low-stearate saturated fat, an unsaturated fat or both (2,4-7,10-13). Other studies have utilized either syn- thetic or randomized stearate-rich fats, but these do not reflect the distinct TAG structures of naturally occurring stearat~rich dietary fats (4,9,16). Lastly, some human studies in metabolic ward settings have used liquid formula diets (3,4,17).

The objective of this work was to gain insight into the modes whereby stearic acid exerts its hypocholesterolemic effect. Three naturally occurring dietary fats, lard (13.2% 18:0), beef tallow (19.2% 18:0), and cocoa butter (34.7% 18:0) were fed to provide graded levels of stearic acid, relatively constant amounts of palmitic acid and distinct TAG pr~ files Cocoa butter is composed chiefly of three TAG species: 1,3~lipalmitoyl-2~leoyl glycerol (POP); 1-palmitoyl-2~leoyl- 3~tearoyl glycerol (POS); and 1,3~listeamyl-2~)leoyl glycerol (SOS). I t has a relatively simple TAG profile compared to beef tallow and lard which are composed of 7-10 TAG species (15,18,19). We compared both fasting and postprandial plasma and hepatic lipid responses, the fat ty acid and TAG profiles of chylomicron (CM) lipids and the levels of hepatic steamyl-CoA and oleoyl-CoA, the substrate and product for the hepatic A9 desaturase system, in rats.

MATERIALS AND METHODS

Animals and diets. Male Sprague-Dawley rats (Harlan Sprague-Dawley, Madison, WI), 150-175 g, were housed in individual stainless-steel cages with free access to water in a room maintained at 25~ using a 12 h reverse light- dark cycle (lights on 2200-1000 h). The animal facilities and protocols were approved by an insti tutional animal care and use commit tee Rats consumed ad libitum one of four diets pat terned after the AIN 76A diet (20), containing 16% fat from either 16% corn oil, or 15% lard, 15% beef tallow or 15% cocoa butter with 1% corn oil to pre- vent essential fa t ty acid deficiency (Table 1). The fat ty acid composition of each diet (Table 2) was determined by capillary gas chromatography with flame-ionization detection (GC-FID). All diets contained 0.2% added cholesterol (Sigma, St. Louis, MO) to provide similar amounts of dietary cholesterol (0.465 mg/kcal). Diets were stored at 4~ and fresh diets were presented to animals biweekly. Food intake was calculated weekly based on a 4-d period and body weight was measured biweekly.

Experimental design. A 4 • 2 (DIET • FED/FASTED) factorial design was used (n = 10). After 3 wk of feeding, all rats were fasted overnight and killed during the early portion of the dark cycle For each of the four diets, one group of animals was killed in a fasted state (FASTED) while the other group was killed in a postprandial state (FED). The FED rats were fed ad libitum for I h and killed 1.5 h later. During the 1 h a d libitum feeding period, animals consumed 6.0 • 0.3 g diet. All animals were anesthetized with CO2 and blood (8-10 mL) was collected by cardiac puncture into syringes containing 1 mg ethy- lenediaminetetraacetic acid and 0.1 mg gentamicin sulfate

Copyright �9 1993 by the American Oil Chemists' Society LIPIDS, Vol. 28, no. 6 (1993)

540

C.C. MONSMA AND D.M. NEY

TABLE 1 TABLE 2

Composition of Stearate-Rich Diets

Diet composition Ingredient a Amount (%)

Casein 20.00 DL-methionine 0.30 Cornstarch 26.89 Sucrose 26.89 Cellulose 5.00 Fat source b 15.00 Corn oil c 1.00 Mineral mix -- AIN 76 3.50 Vitamin mix -- AIN 76A 1.00 Choline bitartrate 0.20 Butylated hydroxytoluene (BHT) 0.02 Cholesterol 0.20 aAU ingredients were obtained from TEKLAD, Harlan Sprague-

Dawley, Inc. (Madison, WI) except for cocoa butter, which was obtained from Ambrosia Chocolate (Milwaukee, WI) and ch~

blesterol and BHT, which were obtained from Sigma (St. Louis, MO). Lard, beef tallow, cocoa butter or corn oil.

CTo provide essential fatty acids.

per mL blood. Plasma was obtained by centrifugation at 4~ for 20 min at 1200 • g. Livers were excised, blot ted dry, weighed and divided into two portions. One port ion was frozen immediately in liquid nitrogen and stored at - 7 0 ~ for the analysis of individual acyl-CoA esters, and the other port ion was stored at - 2 0 ~ for hepatic lipid analyses.

Lipid analyses. For all FED and corn oil FASTED rats, CM, very low density lipoprotein (d < 1.006 g/mL, VLDL), low-density lipoprotein (d = 1.006-1.050 g/mL, LDL) and high-density lipoprotein (d -- 1.050-1.196 g/mL, HDL) were fractionated by sequential ul tracentr ifugation from 3-mL plasma samples containing 0.56 mM, 5,5'-dithi~ bis-nitobenzoic acid and 1 mM phenylmethylsufonylfluor- ide in isopropanol (21). CM were isolated in the top 2 mL after centrifugation for 1.0 h at d < 1.006 g/mL, extracted with hexane]isopropanol (22), evaporated to dryness under nitrogen at 37 ~ C and redissolved in chloroform/methanol (3:2, vol/vol).

Total and free cholesterol were measured in plasma, CM, VLDL, LDL and HDL, and the concentration of esterified cholesterol was determined by difference (21). Trigly- cerides in plasma (Sigma Triglyceride Kit, No. 336) and lipoprotein samples were also measured. Total protein (VLDL, LDL, HDL), phospholipid and H D L apolipopro- tein A-I concentrations were determined as previously described (21). Lipids were extracted from 1-g liver samples with chloroform/methanol (2:1, vol/vol) and assayed for both total and free cholesterol and triglycerides; esterified cholesterol (EC) was determined by difference (21). Individual fa t ty acyl-CoA esters were analyzed by reverse-phase high-performance liquid chromatography (23).

Triglyceride and cholesteryl ester from the CM lipid ex- t rac t were isolated on 3 mL aminopropyl (NH2) Bond Eluts (catalog n a 1212-4038, Varian, Walnut Creek, CA) using a modification of the method of Kalzuny et al. (24). An internal s tandard containing 500 ~g each of trihep- tadecanoin and cholesteryl pentadecanoate (Sigma) was added to a 500 ~L aliquot of CM extract (approximately 1-2 mg total lipid). Triglyceride and cholesteryl ester frac-

Fatty Acid Profiles of Stearate-Rich Diets

Fatty acid Corn oil Lard Beef tallow Cocoa butter (expressed as % methyl esters) a

14:0 ND 1.3 2.9 ND 16:0 11.4 24.9 24.8 25.4 16:1 ND 2.2 2.8 ND 18:0 2.0 13.2 19.2 34.7 18:1 25.6 44.0 44.1 33.5 18:2 60.1 14.2 6.3 6.3 18:3 1.0 ND ND ND 20:4 ND ND ND ND

Ratios 16:0/18:0 5.7 1.9 1.3 0.7 18:0/18:1 0.1 0.3 0.4 1.0 16:0/18:1 0.4 0.6 0.6 0.8

aValues expressed as means for three determinations; ND, not detected.

t ions obtained from the Bond Eluts were evaporated to dryness and stored in chloroformJmethanol (2:1, vol]vol; 250 #L) until analyzed by GC-FID for either fa t ty acid or TAG profiles. This procedure was also used to isolate the triglycerides from the diets for TAG profile analyses.

Fatty acid analyses. Both triglyceride and cholesteryl ester fa t ty acids obtained from CM lipids were transesteri- fled using methanol/benzene (4:1, vol]vol; 2 mL) and acetyl chloride (200 ~L) (25). Fa t ty acid methyl esters (FAME) were stored in benzene at - 2 0 ~ until analysis by capillary GC-FID. Fa t ty acids from the diets were transesteri- fled using 10% BF3/methanol (w/w) reagent (Supelc~ Bellefonte, PA) and stored in pentane at - 2 0 ~ until analyzed. FAME were analyzed on a Varian 3400 gas chromatograph equipped with a flame-ionization detector, a Varian 1093 SPI temperature programmable injector (Palo Alto, CA), a Supelcowax 10 fused silica column, 30 m • 0.32 mm i.d. 0.25 ~m film (Supelco) and an In- Board Data Handling System (IBDH TM, Sugarland, TX). The injector temperature was progammed from 50 to 250~ at a rate of 100~ and held for 15 min during the analysis. The initial column temperature was held for 2 min at 50~ programmed to 160~ at a rate of 30~ then programmed to 190~ at a rate of 3~ and finally programmed to 227 ~ C at a rate of 4~ and held for 15 min for a total analysis t ime of 40 min. The carrier and make-up gases were helium at 2 mL/min and 30 mL/min, respectively. The detector temperature was 300~ Fat ty acids were identified by comparing the retention times with those of known standards (Nu-Chek-Prep, Elysian, MN) and expressed as the weight percent distribution of FAMEs.

TAG profiles. Intact TAG profiles were determined based on total acyl carbon number (C,) where n is the total number of carbon atoms from fa t ty acids esterified to the glycerol backbone Profiles of triglycerides isolated from the diet and composites of CM lipid extract from each FED diet group (n = 8-9) were obtained on a Varian GC-FID equipped with a TAP CB (75% phenyl, 25% methylpolysiloxane) on a WCOT Ultiraetal column, 25 M • 0.25 mm i.d., 0.80 mm o.d., 0.10 ~m film (Chrompack, Raritan, NY). The injector was isothermal at 360~ and injection volume was 0.2 gL of a hexane solution (100 ~L)

LIPIDS, Vol. 28, no. 6 (1993)

STEARATE-RICH DIETARY FATS

541

containing triglycerides obtained from Bond Elut separa- t ion of lipid classes. The initial column tempera ture was 290~ immediate ly p rogrammed to 330~ at a rate of 10~ finally p rogrammed to 355~ at a rate of 2~ and held for 8 rain for a to ta l analysis t ime of 25 rain (18,26,27). The carrier gas was helium at 15 psi column pressure; make-up gas was helium at 22 mL/min. The detector temperature was 370~ Da ta were acquired using a Waters Baseline 810 Chromatography Worksta- t ion connected via a Waters Sys tem Interface Module (Waters, Milford, MA). Manual baseline correction and reintegration was required for all samples due to the com- plexity of the chromatograms. Identif icat ion of TAG species from the dietary fats and CM TAG was based upon compar ison of retention t imes to a triglyceride s tandard (catalog no. 178-11, Sigma) and published chromatograms (18,19). Values are expressed as the area percent of each TAG species.

Statistical analysis. Differences among die tary treat- ment groups were assessed by either one-way or two-way analysis of variance (ANOVA) us ing the SAS general linear models (GLM) program (SAS, Cary, NC) (28). Group means were considered to be significantly different a t P < 0.05 as determined by the protected least significant difference technique. Values are expressed as mean _+ SEM; means with different superscripts are significantly different.

RESULTS

Growth, food intake and liver weight. Final body weight and growth did not differ across the t rea tment groups but diet affected mean daily food intake (Table 3). As the stearic acid content of the diet increased, food intake increased significantly and ra ts fed the cocoa bu t t e r diet ate 10% more than ra ts fed the corn oil diet. Relative liver weight was significantly affected by bo th D I E T and FED/FASTED s ta tes such t ha t F E D animals had 11% higher liver weights than FASTED animals.

Plasma and hepatic cholesterol and triglycerides. Plas- m a cholesterol and triglyceride concentrations did not differ among the groups fed different dietary fats but significant differences occurred between the F E D and FASTED groups (Table 4). F E D groups had 44% greater p la sma triglyceride concentrat ions and 12.5% lower p lasma total cholesterol concentrat ions than FASTED groups. Lower p l a sma cholesterol concentrat ions in F E D animals were associated with significantly lower esterified, but not free, cholesterol levels. The esterified/free cholesterol (E/F) ratio was significantly lower for the three F E D vs. FASTED stearate-rich groups while the corn oil group had similar rat ios in bo th F E D and FASTED states.

In contrast, dietary fat affected hepatic cholesterol and triglyceride concentrat ions more s t rongly than p lasma concentrations and a significant D I E T X FED/FASTED interaction was observed for hepatic total, esterified and E/F cholesterol concentrat ions (Table 5). Compared to fasting, hepatic total and EC decreased after consuming a meal containing corn oil or cocoa butter, showed little change after consuming a meal containing lard and increased after consuming a meal containing beef tallow. Among F E D groups, cocoa but te r showed 54-142% lower (P < 0.001) hepatic total and EC levels than lard and beef tallow. Stearate-rich dietary groups had lower hepatic triglycerides than the corn oil group (corn oil = 15.5; lard, beef tallow and cocoa bu t te r = 6.9-11.0 ~no l triolein/g liver, P = 0.0001, main effects two-way ANOVA).

Hepatic acyl-CoA concentrations. Individual hepatic acyl-CoA concentrat ions in F E D and FASTED animals are summar ized in Table 6. Significant D I E T • FED/FASTED interaction was observed for stearoyl-CoA (18:0). In FASTED animals stearoyl-CoA (18:0) concen- t ra t ions generally reflected 18:0 content of the diet (lard < beef tallow < cocoa butter) but were not significantly different. In contrast , in F E D animals there was no association between dietary 18:0 and stearoyl-CoA levels. M o s t notably , F E D s tea roy l -CoA c o n c e n t r a t i o n s did not differ significantly for corn oil (2.0% 18:0) and

TABLE 3

Final Body Weight, Weight Gain, Liver Weight and Mean Food Intake in Rats Fed Stearate-Rich Dietary Fats for Three Weeks a

Diet

Liver Final body Weight Mean food weight

weight gain intake (g wet tissue/ (g) (g/21 d) (g/d) 100 g body wt)

Fed Corn oil 293 __ 6 113 +- 6 Lard 288 • 6 109 • 6 Beef tallow 295 • 6 115 • 5 Cocoa butter 293 • 6 113 • 5

Fasted Corn oil 281 • 6 102 • 6 Lard 287 • 6 107 • 5 Beef tallow 285 • 6 106 • 5 Cocoa butter 287 • 6 107 +- 5

Two-way ANOVA (GLM), P-values

Diet NS NS Fed/fasted NS NS Diet • fed/fasted NS NS

0 3 c,d 13.2 • 013b,c,d 3.71 • 0.05 a,b 13.5 • 3.65 __ 0.08 a,b 13.9 • 0.3 a'c 3.65 • 0.04 a,b 14.6 • 0.3 a 3.54 + 0.05 a'b'c

13.0 +- 0.3 d 3.41 4- 0.03 b,c,d 13.2 • 0.3 c'd 3.27 • 0.07 c,d 13.8 - 0.3 a'd 3.23 • 0.10 c,d 14.3 - 0.3 a'c 3.20 • 0.05 d

0.0002 0.04 NS 0.0001 NS NS

aValues are means • SEM, n = 9-10. Means with different superscripts in the same column are significantly different [P < 0.05, two, way analysis of variance (ANOVAI, protected least significant difference]; NS, not significant; GLM, general linear models.

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542


TABLE 4

Plasma Cholesterol and Triglyceride Concentrations in Rats Fed Stearate~Rich Dietary Fats for Three Weeks a

Cholesterol

Diet Total Esterified Free E/F ratio Triglycerides

(mmol/L) Fed

Corn oil 1.58 • 0.10 c 1.27 • 0.08 c 0.31 • 0.02 4.1 • 0 1 b'e'd 1.28 • 0.09 a Lard 1.69 • 0.05 b,c 1.33 • 0.04 e 0.36 • 0.02 3.8 • 011 d 1.21 • 0.09 a Beef tallow 1.78 • 0 09 a,b,c 1.38 • 0.08 b,c 0.40 • 0.03 3.7 • 0.3 d 1.31 • 0.07 a Cocoa butter 1.69 • 0105 b,c 1.32 • 0.04 c 0.37 • 0.01 3.6 • 0.1 d 1.30 • 0.06 a

Fasted Corn oil 1.94 + 0.14 a 1.57 +__ 0.10 a 0.37 • 0.05 4.6 • 0.3 a'c 0.92 • 0.02 b Lard 1.89 • 0.07 a,b 1.57 + 0.06 a 0.33 • 0.02 4.8 • 0.2 a 0.88 + 0.04 b Beef tallow 1.86 • 0.06 a,b 1.55 • 0.05 a,b 0.31 • 0.02 5.1 • 0.3 a . 0.86 • 0.03 b Cocoa butter 2.02 • 0.12 a 1.67 + 0.12 a 0.36 + 0.01 4.7 • 0.4 a,b 0.91 • 0.03 b

Two-way ANOVA IGLM), P-values Diet NS NS NS NS NS Fed/fasted 0.0003 0.0001 NS 0.0001 0.0001 Diet X fed/fasted NS NS NS NS NS

aValues are means __ SEM, n = 9-10. Means with different superscripts in the same column are significantly different [P < 0.05, two-way analysis of variance (ANOVA), protected least significant difference]; NS, not significant; E/F ratio, esterified/free; GLM, general linear models.

cocoa b u t t e r (34.7% 18:0), b u t were s i g n i f i c a n t l y h ighe r for an imals fed la rd {13.2% 18:0}. H e p a t i c oleoyl-CoA {18:1) c o n c e n t r a t i o n s were a f fec ted b y D I E T b u t n o t b y F E D / F A S T E D s t a t e s and, in genera l , re f lec ted 18:1 d i e t a r y i n t a k e The beef ta l low d i e t a r y group had s ignif icant- ly h igher hepa t i c oleoyl-CoA concen t r a t ions t h a n the corn oil group.

Lipoprotein concentration and composition. The con- c e n t r a t i o n a n d c o m p o s i t i o n of l i pop ro t e in s d i f fered s ign i f i can t ly a m o n g the fed d i e t a r y t r e a t m e n t groups. In- ges t ion of a mea l con t a in ing cocoa b u t t e r resu l ted in H D L w i t h s i g n i f i c a n t l y more (% b y wt) p ro t e in a n d free cho les te ro l and less E C t h a n H D L f rom a n i m a l s fed beef t a l low or l a rd (Table 7). The cocoa b u t t e r g roup showed

a s i g n i f i c a n t l y lower r a t i o of H D L E / F cho les te ro l t h a n all o t h e r g roups . H D L a p o l i p o p r o t e i n A-I c o n c e n t r a t i o n s d id n o t differ a m o n g the g r o u p s ( d a t a n o t shown}. Taken together , the s ign i f ican t ly lower ra t io of H D L (Triglyceride + EC) /P ro t e in in t he cocoa b u t t e r t h a n t h e beef t a l low a n d la rd g roups s u g g e s t s a g r e a t e r p o p u l a t i o n of l ipid- d e p l e t e d H D L p a r t i c l e s w i t h i n g e s t i o n of cocoa b u t t e r t h a n t h e o t h e r s t e a r a t e - r i c h f a t s s tud ied .

The cocoa b u t t e r group showed s ign i f ican t ly h igher con- c e n t r a t i o n s of L D L prote in , p h o s p h o l i p i d and E C t h a n all o t h e r d i e t a r y g r o u p s which is c o n s i s t e n t w i t h a l a rge r n u m b e r of L D L par t i c l es . A d i f fe ren t t r e n d was seen in VLDL; beef ta l low showed s ign i f ican t ly h igher V L D L protein, e s t e r i f i ed a n d t o t a l cho les te ro l c o n c e n t r a t i o n s a n d

TABLE 5

Hepatic Cholesterol and Triglyceride Concentrations in Rats Fed Stearate-Rich Dietary Fats for Three Weeks a

Cholesterol

Diet Total Esterified Free E/F ratio Triglycerides

(/Lmol/g wet tissue weight) Fed

_ _ 2 2 b,c,d Corn oil 13.0 + 07 b'c 7.9 + 08 d'e 3.89 + 0.20 d,e 1.5 + 02 c'd 12.7 • Lard 16.6 +_ 2"0 a,b 1"9 a,b,c 9.7 + Beef tallow 19.1 ___ 114 a,b 12.7 + 3.15 ___ 0.43 d'e 3.2 • 013 b'c'd _ 117 b'c'd

16.0 • 1"3 a,b 020 b'c'd'e Cocoa butter 10.8 ___ 0.9 c 6.6 • 110 d,e 4.225.13 -+ 0109 a'b'c'd 4.81.6 •177 0"7a0.3 c'd 8.76.0 • 0.6 dl"0b'c'd

Fasted Corn oil 18.0 • 14 a'b 12.6 • 13 b,c 3.26 + 0.69 d,e 2.3 • 0.2 b'c 18.2 • 3.2 a Lard 16.1 _+ 1"4 a,b 12.8 • 1"1 a,b,c 3.75 • 0.10 d,e 2.5 _+ 08 b'c 12.0 • 1.4 b,c,d

1"0 b'c 1"0 b'c 'd 070 a'b'c 0"3 b'c Beef tallow 13.8 • l~0b,c 10.0 • li0d,e 5.72 • 0117a, b,c, d 2.7 __ 0.3c,d 6.2 • 0.3 d Cocoa butter 13.8 • 8.1 • 5.40 • 1.6 • . 7.9 _+ 0.9 c

Two-way ANOVA {GLM), P-values

Diet 0.004 0.0001 0.0001 0.0001 0.0001 Fed/fasted NS NS NS NS NS Diet • fed/fasted 0.0006 0.0001 NS 0.01 NS

aValues are means + SEM, n = 9-10. Means with different superscripts in the same column are significantly different [P < 0.05, two-way analysis of variance (ANOVA), protected least significant difference]; NS, not significant; E/F ratio, esterified/free; GLM, general linear models.

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TABLE 6

Acyl-CoA Levels in Hepatic Tissue from Rats Fed Stearate-Rich Dietary Fats for Three Weeks a

Treatments 6:0 8:0 10:0 12:0 14:0 14:1 16:0 16:1 18:0 18:1 18:2 20:4 Total

(mnol]g wet tissue wt)

Fed Corn oil 2.4 a 1.7 0.5 1.4 a 0.3 d Lard 2.4 a 1.7 0.7 0 8 a'b 1.5 a Beef tallow 2.4 a 1.7 0.6 1"0 a,b 1.5 a Cocoa butter 2.6 a 2.0 0.4 017 b 1.1 a'b

Fasted Corn oil 0.9 b 1.6 0.3 1.4 a 0 5 c'd Lard 1.2 b 1.5 ND 0.6 b 0"9 b,c

1 1 a'b 1"0 a,b Beef tallow 1.2 b 1.5 ND 018a, b li0b Cocoa butter 1.4 b 1.9 ND

Two-way ANOVA (GLM), P-values

Diet NS NS NS 0.004 0.0001 Fed/fasted 0.0001 NS NS NS 0.016 Diet • fed/fasted NS NS NS NS NS Pooled SD 0.82 0.49 0.21 0.55 0.46

0 5 d 3.8 1.6 b 3.0 c 9 I a'b'c 15.4 a 0.5 38.0 1"0 b'c 6.3 3.3 a 5.3 a'b 12:8 a'b 4.7 c 0.7 40.1

10 1 a'b'c 1"2 a,b 4.7 3.0 a 2.5 c 818a, b,c 3.4 c 0.8 31.5 0:5 d 5.7 2.2a, b 3.0 c 3.1 c 0.8 28.7

0.3 d , 3.4 1.5 b 3.6 b'c 5.8 c 10.2 b 0.9 29.5 0 8 c '~ 2 2 a'b 3.8 a'b'c 8.3 b'c l:2a, b,c 5.2 4.5 c 1.0 29.3

6.3 1"7 b 4.8a,b, c 13.3 a 4.9 c 1.0 36.5 2:6a,b 8.7a, b, c 1.6 a 5.8 5.9 a 5.0 c 0.9 34.2

0.0001 NS 0.0185 NS 0.04 0.0001 NS NS NS NS NS 0.04 NS NS NS NS 0.0001 NS NS 0.01 NS 0.004 NS NS 0.43 2.55 1.07 2.56 4.49 3.06 0.51 9.7

aMeans with different superscripts under the same column are significantly different, P < 0.05, n = 7-9; NS, not significant; ND, not detected; ANOVA, analysis of variance; GLM, general linear models.

a s i g n i f i c a n t l y lower p r o p o r t i o n of V L D L t r i g l y c e r i d e s t h a n all o t h e r g r o u p s {data for L D L a n d V L D L n o t shown). CM concen t ra t ions genera l ly ref lected VLDL, and beef t a l low showed s imi la r t r i g lyce r ide s b u t s i gn i f i c an t l y h ighe r c o n c e n t r a t i o n s of t o t a l cho les te ro l t h a n l a rd a n d cocoa bu t t e r .

Fatty acid profiles of CM triglyceride and cholesteryl esters. The f a t t y ac id c o m p o s i t i o n of CM t r i g l y c e r i d e is s u m m a r i z e d in Table 8. Cocoa b u t t e r i n g e s t i o n r e s u l t e d in s i g n i f i c a n t l y g r e a t e r CM t r i g l y c e r i d e 18:0 (% m e t h y l ester) , a n d beef t a l low i n g e s t i o n r e s u l t e d in s i g n i f i c a n t l y g rea te r 18:1 t h a n all o the r diets . The corn oil d i e t a ry group h a d s ign i f i can t ly less 16:0, 18:0 and 18:1 b u t s ign i f i can t ly more 18:2 a n d 20:4 t h a n t h e s t e a r a t e - r i ch d i e t a r y groups . The f a t t y ac id c o m p o s i t i o n of CM c h o l e s t e r y l e s t e r fol- lowed s im i l a r t r e n d s b u t t he p r o p o r t i o n of 18:0 d id n o t d i f fer a m o n g t h e d i e t a r y g roups {data n o t shown). CM c h o l e s t e r y l e s t e r a lso c o n t a i n e d a l a rge {35.6-47.1%) pro- p o r t i o n of a r ach idon ic ac id for al l d i e t a r y groups .

A m o n g the s t ea ra te - r i ch d i e t a ry groups , lower 18:0/18:1 ra t ios were no ted in CM TAG compared to t he correspond-

i ng d ie ts : lard, 0.15 vs. 0.30; beef tallow, 0.16 vs. 0.40; a n d cocoa but ter , 0.35 vs. 1.0, respectively. Regress ion ana lys i s showed a s t r o n g l inea r r e l a t i o n s h i p (r e = 0.985, n = 4, P < 0,0D b e t w e e n t h e 18:0 c o n t e n t of CM t r i g l y c e r i d e s and 18:0 c o n t e n t of t he d i e t s b u t an i n s ign i f i c an t cor- r e l a t ion (r 2 = 0.723, n = 4, P > 0.1) be tween the 18:1 con- t e n t of CM t r i g l y c e r i d e r e l a t i ve to d i e t a r y 18:1 {Fig. 1). T h e p r o p o r t i o n of CM 18:1 was s i g n i f i c a n t l y g r e a t e r in t he beef ta l low t h a n t h e l a rd d i e t a r y group, a l t h o u g h l a rd a n d beef t a l low d i e t s d id n o t d i f fer in t h e p r o p o r t i o n of d i e t a r y 18:1.

TAG profiles of diets and CM based on C,. E a c h d ie t d i s p l a y e d a d i s t i n c t TAG prof i le a cco rd ing to s e p a r a t i o n b y C, {Tables 9 -12 and Figs . 2 a n d 3). Cocoa b u t t e r h a d the s i m p l e s t profile, w i t h on ly t h r e e m a j o r TAG species a t C50, Cs2 a n d Cs4. L a r d a n d beef t a l low TAG c o n t a i n e d p r i m a r i l y C50, C52 and C54, b u t t he degree of u n s a t u r a t i o n was g r e a t l y i nc r ea sed c o m p a r e d to cocoa b u t t e r as in- d i c a t e d b y longe r r e t e n t i o n t i m e s on t h e p o l a r co lumn. Beef t a l low a lso h a d more C48 spec ies t h a n t h e o t h e r s t e a r a t e - r i c h d ie ts .

TABLE 7

Plasma HDL Composition (d = 1.050-1.196 g/mL) in Rats Fed Stearate*Rich Dietary Fats a

Corn oil-fasted Corn oil-fed Lard Beef tallow Cocoa butter Pooled SD

{% by wt)

Protein 36.2 • 0.7 35.0 • 0.6 a 32.9 • 0.6 b 31.5 • 0.6 b 35.4 • 0 6 a 1.9 _ _ _ 017 a'b Phospholipid 16.9 + 0.8 18.4 + 0.6 b 20.3 + 0.7 a'b 21.4 • 0.6 a 20.2 • 2.0

Cholesterol Esterified 22.5 • 0.8 22.3 • 0.7 a 23.6 • 0.7 a 23.0 • 0.7 a 19.9 • 0.7 b 1.9 Free 3.9 __- 0.2 3.1 • 0.2 b 2.6 • 0.2 c 2.4 • 0.2 c 4.6 • 0.2 a 0.5 Total 26.4 • 0.7 25.4 • 0.8 26.2 • 0.6 25.4 • 0.5 24.5 • 0.7 1.8

Triglycerides 20.5 • 1.1 21.2 • 0.9 20.7 • 1.0 21.7 • 0.9 19.9 • 1.0 2.9

(ratio)

E/F cholesterol 3.8 • 0.6 4.3 • 0.2 b 5.5 • 0.3 a 5.9 • 0.4 a 2.6 • 0.2 c 0.8 (TG + EC)/Protein 0.94 • 0.03 1.00 • 0.08 a'b 1.06 • 0.03 a 1.12 • 0.03 a 0.90 • 0.02 b 0.14

avalues are means __ SEM, n = 6-9. Means with different superscripts in the same row are significantly different (P < 0.05, protected least significant difference). E/F, esterified to free ratio; EC, esterified cholesterol; TG, triglycerides; HDL, high-density lipoprotein.

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544


TABLE 8

Fatty Acid Composition of Chylomic~on Triglyceride in Rats Fed Stearate-Rich Dietary Fats for Three Weeks a

Fatty acid Corn oil Lard Beef tallow Cocoa butter Pooled SD

(expressed as % methyl esters)

14:0 0.2 + 0.1 b 0.4 _ 0.2 b 1.8 • 0.1 a 0.4 • 0.1 b 0.3 14:1 0 _ 0 b 0 • 0 b 0.1 • 0.0 a. 0 + 0b 0.1 16:0 13.5 + 0.3 c 24.5 • 0.9 a 24.2 __ 0.5 a,b 22.9 + 0.3 b 2.5 16:1 0.2 +_ 0.1 c 1.1 +_ 0.3 b 2.2 +_ 0.1 a 0.4 +_ 0.1 c 0.4 18:0 2.1 • 0.2 c 7.1 • 0.3 b 8.3 + 0.3 b 16.4 • 1.0 a 1.6 18:1 24.2 • 0.5 c 47.3 __ 1.1 b 51.9 ----- 0.8 a 47.7 + 0.8 b 2.4 18:2 53.8 +_ 0.8 a 16.3 • 0.4 b 9.0 __- 0.4 c 9.5 • 0.2 c 1.4 18:3 0.6 • 0.1 0.6 + 0 4 0.1 + 0.1 0.2 • 0.0 0.6 20:0 0.1 +_ 0.0 b 0 ___ 0 b 0.1 +__ 0.1 b 0.4 • 0.1 a 0.2 20:1 0.3 + 0.1 0.2 _ 0.1 0.2 + 0.1 0.3 • 0~ 0.2 20:2 0.2 • 0.1 a 0.2 • 0.1 a 0.0 -- 0.0 b 0 • 0 0.1 20:3 0.2 • 0.1 a 0 • 0 b 0.0 • 0.0 b 0 • 0 b 0.0 20:4 4.6 • 0.7 a 2.3 • 0.4 b 2.0 • 0.3 b 1.9 _+ 0.3 b 1.3

Ratios 16:0/18:0 6.5 • 0.4 a 3.5 • 0.2 b 2.9 +-- 1.0 b 1.4 • 0.1 c 0.595 18:0/18:1 0.09 • 0.01 c 0.15 • 0.01 b 0.16 • 0.01 b 0.35 _ 0.03 a 0.042

aValues expressed as means • SEM, n = 8-9. Means with different superscripts in the same row are significantly different (P < 0.05, protected least significant difference).

Only the s teara te- r ich fa ts showed changes in the p r o files of CM compared to the TAG profiles of the die ts {Tables 9-12 and Figs. 2 and 3). A m o n g stearate-r ich fats, CM TAG were re la t ive ly enr iched in u n s a t u r a t e d species w i th in a TAG Cn {Tables 10-12 and Figs. 2 and 3), and the e n r i c h m e n t became more p ronounced wi th increased d ie ta ry s tea ra te con ten t . Cocoa b u t t e r CM TAG changed m o s t dramatical ly , wi th the comple te d i sappearance of two ma jo r d ie ta ry TAG species, POS and SOS, a nd for- m a t i o n of several new, more u n s a t u r a t e d species (Fig. 3). Similarly, P O S decreased (but was s t i l l present) in b o t h lard and beef tallow CM TAG while SOS decreased in beef tal low CM TAG (Fig. 2). Bo th lard and beef tal low CM TAG were enr iched in more u n s a t u r a t e d species a t C52 and C54 but , unl ike cocoa but ter , these TAG species were also p re sen t in the d ie t a ry fat.

D I S C U S S I O N

This s t u d y provides new i n f o r ma t i on a bou t the effects associa ted wi th inges t ion of s tear ic acid d u r i n g b o t h f a s t i ng and pos tp r and i a l s ta tes in the rat. We compared c o n s u m p t i o n of three n a t u r a l l y occurr ing, s teara te- r ich d ie t a ry fats p rov id ing graded levels of 18:0 a nd d i s t i n c t TAG profiles. Our da t a provide new evidence t h a t b o t h increased 18:0 con ten t and TAG composi t ion influence abso rp t ion and i n t e s t i na l p rocess ing of 18:0. For example, a TAG species, ident i f ied as POS, was poor ly abso rbed from all s teara te- r ich die ts while ano the r TAG species, identif ied as SOS, was poorly absorbed from bo th the beef tal low and cocoa b u t t e r diets. M a t t s o n e t al. (9) observed t h a t in r a t s fed a series of TAG isomers, 18:0 ester if ied a t e i ther the sn-1 or sn-3 pos i t ion was released as the free acid a nd poor ly absorbed in the presence of ca lc ium and magnesium_ Our CM TAG profile da t a are cons i s t en t wi th

LL

CO

�9 DIET 18:1 rt CMTAG 18:1

6O

50

40

30

20

10

BEEF

o 2 13.2 19.2 34 .7

DIET 18:0 (% FAME)

FIG. 1. Relationship between dietary and chylomicron triacylglyeerol (CM TAG) oleic acid {18:1) as a function of dietary stearie acid {18~). Differences between dietary 18:1 and CM TAG 18:1 increased in pr~ portion to the 18:0 content of the diets. Values are weight percent of fatty acid methyl esters (FAME).

TABLE 9

Comparison of Triacylglyeerol {TAG) Composition of Diet and Chylomicron from Rats Fed a Corn Oil Diet

Diet Chylomicron Peak a TAG b (area %) (area %)

1 C48 ND 1.2 2 (POP) c C50 1.4 3.4 3 (PLP) c 3.4 4.8 4 (PO0) c C52 5.9 5.9 5 (PLO) c 14.7 20.1 6 (PLL) c 18.2 20.5 7 {OOO) c C54 4.2 1.4 8 1.8 1.4 9 (OLO) c 13.9 11.7

10 (OLL) c 19.9 17.8 11 (LLL) c 14.7 8.6

aRefers to order of elution of major chromatographic peaks. bTAG total acyl carbon number. CIdentification based on reference to literature (18,19); ND, not

detected; P, palmitic acid; O, oleic acid; L, linoleic acid.

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545


TABLE I0

Comparison of Triacylglycerol (TAG) Composition of Diet and Chylomicron from Rats Fed a Lard Diet


1 C48 0.4 1.4 2 0.8 1.4 3 (PPS) c C50 1.6 1.9 4 (POP) c 7.6 11.4 5 (PLP) c 4.8 7.4 6 0.6 1.4 7 C52 1.4 0.8 8 (POS) c 22.2 7.3 9 (POO) c 31.1 30.2

10 (PLO) c 10.4 16.5 11 2.1 2.5 12 C54 1.3 0.8 13 (SO0) c 4.1 3.1 14 (OOO + SLS) c 4.1 4.6 15 (SLO) c 2.3 2.4 16 (OLO) c 2.5 3.8 17 1.1 0.8 aRefers to order of elution of major chromatographic peaks. bTAG total acyl carbon number. CIdentification based on reference to literature (18,19); P, palmitic

acid; L, linoleic acid; O, oleic acid; S, stearic acid.

TABLE 12

Comparison of Triacylglycerol (TAG) Composition of Diet and Chylomicron from Rats Fed a Cocoa Butter Diet


1 50 O.4 3.1 2 (POP) c 16.6 9.6 3 1.4 3.2 4 52 ND 2.6 5 ND 13.9 6 (POS) c 41.1 ND 7 2.6 28.2 8 2.6 ND 9 0.5 8.9

1O 54 ND 5.6 11 (SOS) c 27.5 ND 12 3.0 10.1 13 1.5 6.0 14 ND 3.2 15 ND 2.6

aSee Figure 3 for peak identification. bTAG total acyl carbon number. CIdentification based on reference to literature (18,19); ND, not detected; P, pMmltic acid; O, oleic acid; S, stearic acid.

this latter observation because 18:0 is esterified primarily at either sn-1 or sn-3 position in lard, beef tallow and cocoa butter (29), and we observed lower absorption of both SOS and POS species.

Additional evidence for reduced 18:0 absorption is suggested by the observation that rats fed stearate-rich diets showed increased food intake but not increased growth (Table 3). The lower CM than diet TAG 18:0/18:1 ratios, especially in the cocoa but ter dietary group, also suggest reduced absorption of 18:0 relative to 18:1 with greater dietary 18:0 intake (10,12,21). These data are supported by the results of another experiment using diets identical

TABLE 11

Comparison of Triacylglycerol (TAG) Composition of Diet and Chylomicron from Rats Fed a Beef Tallow Diet


1 C48 2.7 1.4 2 4.4 1.8 3 (PPS) c C50 3.7 2.3 4 (POP) c 10.6 10.2 5 5.4 8.6 6 (PSS) c C52 2.9 1.0 7 (POS) c 13.8 6.2 8 (PO0) c 23.5 31.2 9 3.6 11.0

10 (SOS) c C54 4.6 1.5 11 (SOO) c 8.6 4.7 12 (OOO) c 4.3 9.6 13 0.6 2.6

aSee Figure 2 for peak identification. bTAG total acyl carbon number. CIdentification based on reference to literature (18,19); P, pAlmitic

acid; S, stearic acid; O, oleic acid.

to those in the present s tudy where we noted that apparent lipid digestibility decreased with increased dietary 18:0 {30}. Apparent lipid digestibility was: cocoa butter, 78% < beef tallow, 82% < lard, 90% < corn oil, 94% (P < 0.001).

Other explanations for the lower 18:0/18:1 ratios observed in CM TAG relative to corresponding diets include conversion of dietary 18:0 to 18:1 by intest inal A9 desaturase and the contribution of endogenous fa t ty acids by the intestine prior to reassembly of CM TAG {31,32}. Giron e t aL {31) observed higher A9 desaturase activity in duodenal mucosa than in the liver. Perhaps the small intestine plays a regulatory role in fa t ty acid metabolism by modifying the degree of saturation of dietary fa t ty acids and TAG structure prior to CM transport out of the enterocyte~

The greater sensitivity of hepatic than plasma chc, lesterol concentrations to dietary fat was due to differences in fed groups. Differences in the FED response li.e., increased or decreased hepatic cholesterol concentrations after a meal} to dietary stearate-rich fats emphasize the important role of the intestine in cholesterol metabolism. Among fed rats, hepatic total cholesterol concentrations were 54-77% lower in the cocoa butter than the lard and beef tallow dietary groups. These data support a liver cholesterol-lowering effect of cocoa but ter compared to beef tallow and lard.

In general, our research confirms previous observations of differences in hepatic but not plasma total cholesterol concentrations in rats fed s tearat~rich vs. unsaturated dietary fats and sampled after an overnight fast {6,7,21}. A longer feeding period and/or larger amounts of dietary fat, cholesterol and cholic acid may be needed to detect differences in plasma cholesterol given the rats ' ability to regulate plasma cholesterol concentrations. For example, Morrissey e t al. (33) noted that 60 d of feeding 10, 20 or 30% cocoa butter diets to male Sprague-Dawley rats

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546


A

r r t- O {3. ( D o

E3

COCOA BUTTER 6

\

11

2 13

=C50 ! C52 ' C54'

B

o

0

.......

I'0 I'5 2'0

\

k,

12

lOI,

IC48= 'C50 C52 C54

1'0 2'O Time (Minutes)

FIG. 2. A: Gas-liquid chromatogram of intact triacylglyeerol (TAG) species according to total acyl carbon number (C46-C54) from a 15% beef tallow + 1% corn oil diet. Column" TAP CB on WCOT Ultimetal (25 m X 0.25 mm, i.d.). Temperature: 290~ immediately programmed to 330~ at 10~ then programmed to 355~ at 2~ and held for 8 min. Carrier gas: helium (15 psi). Peak identification: 1,2. unknown; 3. PSS; 4. POS; 5. unknown; 6. PSS; 7. POS; 8. POO; 9. unknown; 10. SOS; 11. SO0; 12. OOO; and 13. unknown; where P, puimitic acid; O, oleic acid; L, linolcic acid; and S, stearic acid. B: Gas-liquid chromatogram of intact TAG species from chylomierons in rats fed a 15% beef tallow + 1% corn oil diet. Refer to above for peak identification (Refs. 18,19). See Table 11 for area percent.

was needed to detect a significant decrease in plasma cholesterol concentrations compared to corn oil.

Hepatic concentrations of stearoyl-CoA and oleoyl-CoA provide indirect evidence that little hepatic conversion of 18:0 to 18:1 occurred during feeding of stearatc=rich diets (Table 6). Stearoyl-CoA and oleoyl-CoA concentrations were not significantly different for corn oil (2.0% 18:0)

A

K/) t-" O Q. O9

ID (D a

BEEF TALLOW

ii ! !! 'LC52 " C46 C48 C50 C54

11

!

B

r c- o E2. (/)

4.-* (D a

1'0 l'S

4

' \ ' ~ 1.2 1 ~

I �9 m

'C46"C48 C50 C52 C54

3

1'o l'S Time (Minutes)

FIG. 3. A: Gas-liquid chromatogram of intact triacylglycerol (TAG) species from a 15% cocoa butter -t- 1% corn oil diet. Peak identification: 1. unknown; 2. POP; 3. nnknown; 6. POS; 7-9. , ,known; 11. SOS; and 12,13. unknown. B: Gas-liquid chromatogram of intact TAG species from chylomicrons in rats fed a 15% cocoa butter -I- 1% corn oil diet. Peak identification (Refs. 18,19): 1. unknown; 2. POP; 3-5,7,9,10,12-15. unknown. See Table 12 for area percent.

and cocoa butter (34.4% 18:0) dietary groups during feeding. If hepatic A9 desaturase was rapidly converting dietary 18:0 to 18:1, a higher concentration of hepatic stearoyl-CoA and oleoyl-CoA would be predicted for the cocoa butter than for the corn oil dietary group. Hepatic A9 desaturase has been well studied in rats and mice (34-36). Ntambi (36) noted that diets supplemented with essential fatty acids repressed induction of A9 desaturase mRNA in mouse liver following fasting and refeeding.

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547


Since our diets con ta ined 1% corn oil, th is m a y explain the appa ren t lack of hepat ic A9 desa tu rase ac t iv i ty sugges ted by hepat ic oleoyl-CoA concentra t ions .

I n conclusion, this s t u d y demons t ra tes t h a t bo th stearic acid content and TAG composi t ion of stearate-rich dietary fa ts influence intest inal process ing and pos tprandia l hepat ic and l ipoprotein cholesterol concen t ra t ions in the rat. Our d a t a are cons is ten t wi th reduced absorp t ion of stear- a te f rom na tu ra l ly ocur r ing s tearate-r ich d ie ta ry fats. Hepa t i c s tearoyl-CoA and oleoyl-CoA concen t ra t ions do no t sugges t rapid convers ion of d ie ta ry s teara te to oleate dur ing feeding. Fu r the r research on intest inal A9 desaturase and the possible r egu la to ry role of the in tes t ine m a y provide useful ins igh t in to the effects associa ted wi th inges t ion of s tearate-r ich d ie ta ry fats. Given the d i s t inc t TAG profiles of na tu ra l ly occur r ing stearatc~rich d ie ta ry fats, i t m a y no t be p r u d e n t to make general ized state- m e n t s abou t these fa ts based on s tear ic acid con ten t a lone

ACKNOWLEDGMENTS This research was supported by a grant from the National Live Stock and Meat Board and by funds from the College of Agricultural and Life Sciences, University of Wisconsin-Madison, project Na 3096. The authors would like to thank Ambrosia Chocolate, Milwaukee, WI, for their donation of cocoa butter. The technical assistance provided by Mike Grahn and the support of Dr. Rhona Applebaum of the Chocolate Manufacturers Association are greatly appreciate&

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[Received August 20, 1992, and in revised form March 12, 1993; Revision accepted April 6, 1993]

LIPIDS, Vol. 28, no. 6 (1993)

Documents

Interrelationship of stearic acid content and triacylglycerol composition of lard, beef tallow and cocoa butter in rats