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Blood plasma lipoprotein and tissue cholesterol of calves fed soybean oil, corn oil, vegetable shortening or tallow
513
Atherosclerosis, 37 (1980) 513-520 0 Elsevier/North-Holland Scientific Publishers, Ltd
BLOOD PLASMA LIPOPROTEIN AND TISSUE CHOLESTEROL OF CALVES FED SOYBEAN OIL, CORN OIL, VEGETABLE SHORTENING OR TALLOW
MARLENE J. RICHARD, JEANNE W. STEWART, THOMAS R. HEEG, KENNETH D. WIGGERS and NORMAN L. JACOBSON Nutritional Physiology Group, Animal Science Department, IA 50011 (U.S.A.)
Iowa State University, Ames,
(Received 22 February, 1980) (Revised, received 11 August, 1980) (Accepted 13 August, 1980)
Summary The objective of this study was to determine cholesterol content of blood plasma, blood plasma lipoproteins and tissues of calves fed fats of differing compositions. Groups of Z-week-old calves were fed one of the following fats in a reconstituted milk formula: soybean oil, corn oil, vegetable shortening or tallow. The diets contained no dry feed or added cholesterol. Blood plasma cholesterol concentrations increased with time for all groups. After 15 weeks, cholesterol concentrations were greater in the blood, liver and fat of the groups fed soybean oil and corn oil than in those of the groups fed vegetable shortening and tallow. Low density lipoprotein was identified as the carrier of the increased amounts of cholesterol noted in the blood. Key words:
Calf - Corn oil - Lipoprotein cholesterol - Plasma cholesterol oil - Tallow - Tissue cholesterol - Vegetable shortening
- Soybean
Introduction Blood plasma cholesterol concentrations of calves consuming reconstituted milk diets with soybean oil (SBO) providing 30% of the calories are greater than those of calves fed similar tallow (T) diets [l--4]. Also, certain tissues This work was supported in part by funds provided by the Iowa Beef Industry Council, Ames, IA. Journal Paper No. J-9624 of the Iowa Agriculture and Home Economics Experiment. Station, Ames, IA. Project 2184. Correspondence to: Marlene J. Richard, 313 KiIdee Ha& Iowa State University, Ames, IA 50011. U.S.A.
514
from SBO-fed calves contain a larger concentration of cholesterol than those from the T-fed animals. The present study evaluated 2 additional fats, corn oil (CO) and vegetable shortening (VS). Corn oil, a fat similar to SBO in fatty acid composition except for a lower linolenic acid content, was fed to determine if dietary linolenic acid might be an agent in the unusual plasma cholesterol response of calves to unsaturated fat intake (increased plasma cholesterol). Because SBO and T are quite different in degree of saturation, a partially hydrogenated vegetable oil product, VS, was fed to determine if an intermediate plasma cholesterol concentration would result. Lipoprotein cholesterol determinations are important in the diagnosis of human susceptibility to atherosclerosis because high density lipoprotein cholesterol (HDL-C) has been negatively correlated and low density lipoprotein cholesterol (LDL-C) has been positively correlated with this disease [5]. The calf lipoprotein pattern (more HDL than LDL) is unlike that of the human. A dietary effect causing worsening of favorable lipoprotein composition in the calf might help delineate factors creating the unfavorable pattern prevalent in atherosclerosis-prone humans. Materials and Methods Animals
and management
Twenty milk-fed male Holstein calves 2 weeks of age were divided into 4 comparable groups and for 15 weeks were fed a reconstituted milk containing 12% nonfat dry milk solids l and one of the following fats: SBO (“Edsoy” soybean oil) 2, CO (corn oil) 2, VS (“Crisco” vegetable shortening) 3 or T (beef tallow) 4. Each of these fats was incorporated at a level of 2% into reconstituted nonfat milk, employing a Gaulin homogenizer 5. Fat globule size was comparable to that of cow’s milk. Nipple pail feedings were made twice daily at the rate of 100 g milk daily/kg body weight. All diets were supplemented with vitamins 6q7 and minerals 7 at the level recommended by the National Research Council [6] and 0.4 mg chlortetracycline * daily/kg body weight. The calves were penned individually and bedded on wood shavings. On the day of diet initiation and at weekly intervals thereafter, blood samples (fasting) were removed 12 h after feeding and body weights were’determined. At the end of the experiment (15 weeks), the calves were continued on the diets for l-3 more day until slaughter and removal of samples of liver, biceps femoris muscle and perirenal fat. Analytical
procedures
Blood plasma (from blood to which 10 mg EDTA was added per 10 ml of 1 2 3 4 5 6 7
Associated Milk Producers Inc., Mason City, IA, Low Heat Grade A. Staley Mfg. Co., Decatur, IL, courtesy of K. Wright. Proctor and Gamble, Cincinnati, OH. Iowa State University Meat Laboratory, Ames. IA. Gaulin Corp.. Everett, MA. Hoffman-LaRoche Inc.. Nutley, NJ, vitamins A, D and E through the courtesy of G. Landau. Feed Specialties Co., Des Moines, IA, B-complex vitamins and minerals through the courtesy Vohs. 8 Diamond Shamrock Chemical Co., Newark, NJ.
of R.
515
blood) was frozen and stored at -25°C until analysis for total cholesterol by an automated calorimetric procedure [ 71. All tissue samples were freeze-dried and then stored at 4”C. The dry liver and muscle tissues were ground in a Wiley mill. Duplicate l-g samples of each tissue were extracted with 50 ml isopropan01 for 24 h on a wrist-action shaker, then re-extracted with 25 ml isopropanol for 24 h. Adipose samples were kept cold while each l-g sample was minced with surgical scissors as it was added to the isopropanol. (Adipose tissue from animals fed unsaturated fats is very soft at room temperature.) Adipose sample residues from 1st extraction were macerated in the solvent of the 2nd extraction before being shaken. The 2 extracts of each sample were filtered through glass wool, combined and diluted as necessary to permit cholesterol analysis by the same method used for the blood extracts [7]. Plasma to be used for the ultracentrifugal lipoprotein fractionation was stored at 4°C until analysis. The lipoprotein classes separated in this study were: chylomicrons + very low density lipoproteins (VLDL), d < 1.006; low density lipoproteins (LDL), d 1.006-1.063; high density lipoproteins (HDL), d 1.063-1.21; and very high density lipoproteins (VHDL), d > 1.21. Each lipoprotein class was obtained by ultracentrifugal flotation above successively increased densities according to Have1 et al. [8]. The lipoprotein samples were stored at -25°C. Total cholesterol in each fraction was assayed by the cholesterol esterase-cholesterol oxidase method [ 91. Clean separation of the lipoprotein classes was confirmed with polyacrylamide gel disc electrophoresis
[lOI * Statistical analysis A split-plot design was employed. The data were evaluated by analysis of variance [ll]. The pooled variation among animals treated alike was used to test the significance of the effect of dietary fat on cholesterol response. The effects of repeated measurements on the same animals across the 15-week period of the experiment and the diet-by-week interaction were tested for significance by the residual error. Three comparisons were made: first, the data of the 2 groups of calves fed the less-saturated fats (SBO + CO) were compared with those of calves fed the more-saturated fats (VS + T); then data from calves fed SBO were compared with those of calves fed CO; finally, similar comparisons were made between the VS- and T-fed calves. Further analyses of pair treatment comparisons were calculated by least significant difference [ 121. Results The calf growth rate, calculated as percentage gain from 0 to 15 weeks, did not vary among dietary groups (Table l), nor were average weekly weights significantly different (Table 2). Plasma cholesterol concentrations increased as the experiment progressed for all 4 groups (P < 0.005). At week 0 plasma cholesterol in mg/dl for SBO, CO, VS and T groups was 90, 91, 108 and 72, respectively; at week 15 the respective values were 161, 141, 113 and 111. Analysis of variance revealed no significant differences in plasma cholesterol concentrations between the 2 unsaturated fats, SBO and CO, nor between the more saturated pair, VS and T. The sum of the plasma cholesterol concentra-
516 TABLE
1
AVERAGE Dietary
BODY group
Soybean Corn
WEIGHT
GAINS Initial
a
oil
oil
Vegetable
shortening
TZdlOW a Five
calves
Final
weight
15-wk
weight
(kg)
(kg)
(%)
43.3
90.5
109
42.6
90.3
112
44.7
94.8
112
37.5
79.1
111
gain
per group.
tion of the groups fed unsaturated fats was significantly different (P < 0.001) from that of the 2 groups fed more saturated fats (Fig. 1 and Table 2). A least significant difference test of all individual pairs showed that both SBO and CO were different from each of the more saturated fats, VS and T (P < 0.01). Statistical analysis of lipoprotein cholesterol revealed that while cholesterol in VLDL, HDL and VHDL was unaffected by diet, cholesterol in LDL was elevated to a greater degree by the SBO and CO diets (Fig. 2 and Table 2) than by the VS and T diets. Cholesterol concentration was greater in the liver (P < 0.001) and fat (P < 0.05) tissue of calves fed SBO and CO than in tissues of calves fed the other diets; muscle concentration was not affected by diet (Table 3). Cholesterol concentration of the liver of the CO group was significantly different than that of the VS and T groups when tested individually by least significant difference (P < 0.01). While the cholesterol concentrations of liver, muscle and fat tissues of the SBO group were not significantly different from those of VS or T
TABLE
2
ANALYSIS
OF
VARIANCE
OF
BLOOD
PLASMA
COMPONENTS
AND
AVERAGE
WEEKLY
WEIGHTS Source
of variation
Plasma
cholesterol
Average
weekly
weight Total
In LDL
In HDL F
df df
F
df
F
F
Treatment (Trt) SBO + CO YS VS+T SBO
vs CO
VSvsT Animals
(within
3
11.27
a
3
1
31.89
a
Weeks
X Trt
Residual
error
1.36
3
1.83
a
1
26.11
0.76
1
0.49
1.34
1
0.08
1.80
1
0.06
1
0.59
1
0.26
1.51
1
(6915)
15d
Weeks
a
1
16
Trt)
8.82
C
12.47
45
d
240
d
2.73
16
(1746)
(103)
23.41
a
9.61
a
15
9
5.73
a
2.27
b
45
48
(133)
(448)
P
in parentheses
conservative
P
df.
are mean i.e.,
weeks
squares
196
3
f
a
d Used
(1675)
e
(443)
for
= 1, weeks
error X Trt
used
to test
= 3, residual
significance. error
= 16.
4.94
16
240
a
0.51 (24)
b
PLASMA
__J
\ 60.
CHOLESTEROL
‘\ :/ 0
,I”
2
4
6
6
IO
12
14
WEEKS Fig. 1. Mean blood plasma cholesterol at weekly intervals (5 calves per group). soybean oil, CO = corn oil, VS = vegetable shortening. and T = tallow.
groups, the mean liver cholesterol concentration calves than in VS or T calves (Table 3).
was slightly
Dietary
groups;
greater
SBO =
in SBO
Discussion The observation that the bovine species is subject to spontaneous atherosclerosis [13-161 and to cholesterol-induced atherogenic changes in the aorta LIPOPROTEIN
CHOLESTEROL
CONCENTRATION
VHDL __-.---------______.-,~.-,~, VLDL
VEG. SHORTENING
I
._.------I------_____
0
___,.,
5
I
VHDL i6vLDL
---_.,.. _______ ‘.j; .__________..VHDL “lo’. ,_
IO
15”mL TIME (WEEKS)
Fig. 2. Mean cholesterol
in various lipoproteins
as mg/dl of blood Plasma (5 cakes
Per group).
518 TABLE
3
MEANS
AND
Dietary
group
Soybean
oil
corn
STANDARD
ERRORS
a
TISSUE
CHOLESTEROL
Liver
oil
Vegetable
OF
b
mg dry
Adipose
1.39
? 0.09
0.30
+ 0.01
0.41
f 0.06
+ 0.11
0.30
+ 0.01
0.37
f 0.04 k 0.02
1.16
?r 0.06
0.29
f 0.01
0.27
1.15
+ 0.07
0.29
? 0.01
0.32
SBO+COvsVS+T
P <
SBO
vs CO
VSvsT a Five
calves
b Biceps
0.001
Nsd
P <
NS
NS
NS
NS
NS
NS
tissue)
tissue
1.55
W
Tall0
shortening
Muscle
@g/100
c
+ 0.05 0.05.
per group.
femoris.
c Perirenal. d NS
= not
significant.
and coronary arteries [17,18] led to the study of effect of feeding saturated and unsaturated fats with and without cholesterol to nonruminating calves [ 191. Calves consuming SBO as 30% of their dietary calories, a level similar to that recommended for humans, have greater plasma cholesterol concentrations than do calves consuming T in the same manner [l-4]. Differences in tissue cholesterol concentrations in liver, fat and muscle were observed, with the SBO-fed calves usually having higher concentrations. Accumulation of tissue cholesterol in response to unsaturated fat feeding has been observed also in growing lambs [20] and rats [21]. In this experiment, there were no cholesterol concentration distribution differences noted between CO- or SBO-fed calves. CO was fed to compare an unsaturated fat that contained by our analysis no detectable linolenic acid with SBO, which contained about 5% linolenic acid (Table 4). Vegetable shortening was fed to ascertain the effect of partial hydrogenation of SBO on the cholesterol distribution in the calves. Although VS contained a lower percentage of saturated fatty acids than T (Table 4), the results from feedings VS were remarkably like those of feeding T. High density lipoproteins constitute about 80% of all adult bovine serum lipoproteins [22,23]; similar observations have been made for the plasma of TABLE
4
LONG
CHAIN
Fatty 14:o
FATTY
acid
ACID Soybean
DISTRIBUTION
IN Corn
oil
DIETARY oil
FATS
a
Vegetable
Tallow
shortening
b
5
16:0
15
15
16
37
12
11 42
16:l
5
18:0
4
1
18:l
25
26
46
18:2
51
58
25
‘18:3
5
a Methyl
esters
&co1
succinate
b Carbon
number:
of
the
1
fatty
column number
acids
were
at 180°C,
and
of double
separated each
bonds.
by
is reported
gas chromatography as a percentage
employing of total
fatty
a 20% acid.
diethylene
519
young calves [24]. At the start of this experiment, two-thirds of the total plasma cholesterol of the calves was found in the HDL fraction. By the end of the experimental period, the situation was reversed for the SBO and CO groups of calves. Two-thirds of the total plasma cholesterol was found in the LDL fraction of these groups; at this point, the plasma cholesterol of the VS and T groups of calves was equally split between the LDL and HDL fractions. When Dryden et al. [25] compared ruminating steers consuming untreated safflower oil with steers consuming safflower oil which was treated to be nonfermentable in the rumen, they found a 3-fold increase in the serum LDL concentration of those consuming the treated oil. At the end of the current experiment with nonruminating calves, a 3-fold increase above initial concentration in LDL cholesterol was observed in SBO and CO groups and a l-fold increase above initial concentration in LDL cholesterol was observed in VS and T groups. At week 0 of our experiment, the average ratio of LDL-C to HDL-C in all calf plasma was 0.46; at week 15, the same ratio was 2.3 for the SBO group, 2.2 for the CO group, 1.5 for the VS group and 1.3 for the T group. Although there has been disagreement among researchers as to the significance of LDL-C/ HDL-C changes, a 1979 Framingham Study perspective [5] states, “A relatively large amount of cholesterol in the lowdensity lipoprotein fraction is atherogenie, whereas that in the high-density fraction appears protective”. Our studies demonstrate clearly that dietary SBO and CO greatly increase LDL-C in the nonruminating calf, the mechanism of which has yet to be explained. Acknowledgements The authors wish to thank Dr. D.F. Cox for guidance in the statistical analyses, E.P. Vining for assistance with the enzymatic cholesterol assays and L.E. Sampson for assistance in the preparation of the manuscript. References 1 Wiggers, K.D., Richard, M.J., Stewart, J.W., Jacobson, N.L. and Berger, P.J., Type and amount of dietary fat affect relative concentration of cholesterol in blood and other tissues of calves, Lipids, 12 (1977) 586. 2 Heeg. T.R.. The Effects of Tallow or Soybean Oil Reconstituted Milk Diets on Plasma and Tissue Cholesterol in Calves, MS. Thesis, Iowa State University Library, Ames, IA, 1977. 3 Barrows, K.K.. Heeg, T.R.. McGilIiard, A.D., Richard, M.J. and Jacobson, N.L., Effect of type of dietary fat on plasma and tissue cholesterol of calves, J. Nutz., 110 (1980) 335. 4 Stewart, J.W., Wiggers. K.D.. Jacobson, N.L. and Berger, P.J.. Effect of various triglycerides on blood and tissue cholesterol of calves, J. Nutr., 108 (1978) 561. 5 Kannel. W.B., Castelli, W.P. and Gordon, T., New perspectives based on the Framingham study, Ann. Intern. Med., 90 (1979) 85. 6 Jacobson, N.L., Bath, D.L., Miller, W.J., Moe, P.W.. Reid, J.T., Schultz, L.H. and Swanson, E.W., Nutrient Requirements of Dairy Cattle, National Academ; of Sciences, National Research Council, Washington, DC, 1978. 7 Technicon Auto-Analyzer Methodology N-24A, Total cholesterol in serum, Technicon Instruments Corp.. Chauncey, NY, 1965. 8 Havel, R.J., Eder. H.A. and Bragdon, J.H., The distribution and chemical composition of ul&racentrifugally separated lipoproteins in human serum, J. Clin. Invest., 34 (1955) 1345. 9 Allain, C.C.. Peon, L.S., Ghan, C.S.G., Richmond, W. and Fu, P.. The enzymatic determination of total serum cholesterol. Clin. Chem.. 20 (1974) 470. 10 Naito, H.K., Wada, M., Ehrhart, L.A. and Lewis, L.A., Polyacrylamide-gel disc-electrophoresis as a screening procedure for serum lipoprotein abnormslities, Clin. Chem.. 19 (1973) 228.
520 11 12 13 14 15 16 17 18 19
20 21 22 23
24 25
Snedecor. G.W. and Cochran, W.G.. Statistical Methods, Iowa State University Press, Ames, IA, 1967. Cochran, W.G. and Cox. G.M.. Experimental Designs, WiIey and Sons, New York, NY 1957. Stehbens, W.E. InthnaI proliferation and spontaneous lipid deposition in the cerebral arteries of sheep and steers, J. AtheroscIer. Res.. 5 (1965) 556. Likar, I.N. and Robinson, R.W., Bovine arterial disease, Part 1 (Localization of lipids in the abdominal aorta in relation to bovine atherosclerosis). Arch. Path., 82 (1966) 555. Skold, B.H.. Jacobson, N.L. and Getty, R., Spontaneous atherosclerosis of bovine, J. Dairy Sci., 50 (1967) 1712. Wiggers, K.D., Jacobson, N.L. and Getty. R., Atherosclerosis in ruminants, J. Anim. Sci.. 32 (1971) 1037. atherosclerosis in the young bovine, Wiggers, K.D.. Jacobson. N.L. and Getty. R., Experimental Atherosclerosis, 14 (1971) 379. Wiggers, K.D.. Jacobson, N.L.. Getty, R. and Richard, M., Mode of cholesterol ingestion and atheros clerosis in the young bovine, Atherosclerosis, 17 (1973) 281. Jacobson, N.L., Richard, M., Berger, P.J. and KIuge. J.P.. Comparative effects of tallow, lard and soybean oil, with and without supplemental cholesterol, on growth, tissue cholesterol and other responses of calves, J. Nutr., 104 (1974) 573. Kirk, R.D.. Elevated cholesterol concentration in muscle of lambs fed unsaturated fats, N.Z. Med. J.. 82 (1975) 58. Angel, A. and Farkas, J., Regulation of cholesterol storage in adipose tissue. J. Lipid Res., 15 (1974) 491. Jonas, A., Physicochemical properties of bovine serum high density lipoprotein, J. Biol. Chem., 247 (1972) 7767. Dryden, F.D., Marcello. J.A., Adams, G.H. and Hale, W.H.. Bovine serum lipids, Part 2 (Lipoprotein quantitative and qualitative composition as influenced by added animal fat diets), J. Anim. Sci., 32 (1971) 1016. Richard, M.J., Unpublished observations (1979). Dryden, F.D., Marcello, J.A.. Cuitun, L.L. and Hale, W.H.. Protein protected fat for ruminants, Part 2 (Serum lipids and lipoproteins), J. Anim. Sci., 40 (1975) 697.
Atherosclerosis, 37 (1980) 513-520 0 Elsevier/North-Holland Scientific Publishers, Ltd
BLOOD PLASMA LIPOPROTEIN AND TISSUE CHOLESTEROL OF CALVES FED SOYBEAN OIL, CORN OIL, VEGETABLE SHORTENING OR TALLOW
MARLENE J. RICHARD, JEANNE W. STEWART, THOMAS R. HEEG, KENNETH D. WIGGERS and NORMAN L. JACOBSON Nutritional Physiology Group, Animal Science Department, IA 50011 (U.S.A.)
Iowa State University, Ames,
(Received 22 February, 1980) (Revised, received 11 August, 1980) (Accepted 13 August, 1980)
Summary The objective of this study was to determine cholesterol content of blood plasma, blood plasma lipoproteins and tissues of calves fed fats of differing compositions. Groups of Z-week-old calves were fed one of the following fats in a reconstituted milk formula: soybean oil, corn oil, vegetable shortening or tallow. The diets contained no dry feed or added cholesterol. Blood plasma cholesterol concentrations increased with time for all groups. After 15 weeks, cholesterol concentrations were greater in the blood, liver and fat of the groups fed soybean oil and corn oil than in those of the groups fed vegetable shortening and tallow. Low density lipoprotein was identified as the carrier of the increased amounts of cholesterol noted in the blood. Key words:
Calf - Corn oil - Lipoprotein cholesterol - Plasma cholesterol oil - Tallow - Tissue cholesterol - Vegetable shortening
- Soybean
Introduction Blood plasma cholesterol concentrations of calves consuming reconstituted milk diets with soybean oil (SBO) providing 30% of the calories are greater than those of calves fed similar tallow (T) diets [l--4]. Also, certain tissues This work was supported in part by funds provided by the Iowa Beef Industry Council, Ames, IA. Journal Paper No. J-9624 of the Iowa Agriculture and Home Economics Experiment. Station, Ames, IA. Project 2184. Correspondence to: Marlene J. Richard, 313 KiIdee Ha& Iowa State University, Ames, IA 50011. U.S.A.
514
from SBO-fed calves contain a larger concentration of cholesterol than those from the T-fed animals. The present study evaluated 2 additional fats, corn oil (CO) and vegetable shortening (VS). Corn oil, a fat similar to SBO in fatty acid composition except for a lower linolenic acid content, was fed to determine if dietary linolenic acid might be an agent in the unusual plasma cholesterol response of calves to unsaturated fat intake (increased plasma cholesterol). Because SBO and T are quite different in degree of saturation, a partially hydrogenated vegetable oil product, VS, was fed to determine if an intermediate plasma cholesterol concentration would result. Lipoprotein cholesterol determinations are important in the diagnosis of human susceptibility to atherosclerosis because high density lipoprotein cholesterol (HDL-C) has been negatively correlated and low density lipoprotein cholesterol (LDL-C) has been positively correlated with this disease [5]. The calf lipoprotein pattern (more HDL than LDL) is unlike that of the human. A dietary effect causing worsening of favorable lipoprotein composition in the calf might help delineate factors creating the unfavorable pattern prevalent in atherosclerosis-prone humans. Materials and Methods Animals
and management
Twenty milk-fed male Holstein calves 2 weeks of age were divided into 4 comparable groups and for 15 weeks were fed a reconstituted milk containing 12% nonfat dry milk solids l and one of the following fats: SBO (“Edsoy” soybean oil) 2, CO (corn oil) 2, VS (“Crisco” vegetable shortening) 3 or T (beef tallow) 4. Each of these fats was incorporated at a level of 2% into reconstituted nonfat milk, employing a Gaulin homogenizer 5. Fat globule size was comparable to that of cow’s milk. Nipple pail feedings were made twice daily at the rate of 100 g milk daily/kg body weight. All diets were supplemented with vitamins 6q7 and minerals 7 at the level recommended by the National Research Council [6] and 0.4 mg chlortetracycline * daily/kg body weight. The calves were penned individually and bedded on wood shavings. On the day of diet initiation and at weekly intervals thereafter, blood samples (fasting) were removed 12 h after feeding and body weights were’determined. At the end of the experiment (15 weeks), the calves were continued on the diets for l-3 more day until slaughter and removal of samples of liver, biceps femoris muscle and perirenal fat. Analytical
procedures
Blood plasma (from blood to which 10 mg EDTA was added per 10 ml of 1 2 3 4 5 6 7
Associated Milk Producers Inc., Mason City, IA, Low Heat Grade A. Staley Mfg. Co., Decatur, IL, courtesy of K. Wright. Proctor and Gamble, Cincinnati, OH. Iowa State University Meat Laboratory, Ames. IA. Gaulin Corp.. Everett, MA. Hoffman-LaRoche Inc.. Nutley, NJ, vitamins A, D and E through the courtesy of G. Landau. Feed Specialties Co., Des Moines, IA, B-complex vitamins and minerals through the courtesy Vohs. 8 Diamond Shamrock Chemical Co., Newark, NJ.
of R.
515
blood) was frozen and stored at -25°C until analysis for total cholesterol by an automated calorimetric procedure [ 71. All tissue samples were freeze-dried and then stored at 4”C. The dry liver and muscle tissues were ground in a Wiley mill. Duplicate l-g samples of each tissue were extracted with 50 ml isopropan01 for 24 h on a wrist-action shaker, then re-extracted with 25 ml isopropanol for 24 h. Adipose samples were kept cold while each l-g sample was minced with surgical scissors as it was added to the isopropanol. (Adipose tissue from animals fed unsaturated fats is very soft at room temperature.) Adipose sample residues from 1st extraction were macerated in the solvent of the 2nd extraction before being shaken. The 2 extracts of each sample were filtered through glass wool, combined and diluted as necessary to permit cholesterol analysis by the same method used for the blood extracts [7]. Plasma to be used for the ultracentrifugal lipoprotein fractionation was stored at 4°C until analysis. The lipoprotein classes separated in this study were: chylomicrons + very low density lipoproteins (VLDL), d < 1.006; low density lipoproteins (LDL), d 1.006-1.063; high density lipoproteins (HDL), d 1.063-1.21; and very high density lipoproteins (VHDL), d > 1.21. Each lipoprotein class was obtained by ultracentrifugal flotation above successively increased densities according to Have1 et al. [8]. The lipoprotein samples were stored at -25°C. Total cholesterol in each fraction was assayed by the cholesterol esterase-cholesterol oxidase method [ 91. Clean separation of the lipoprotein classes was confirmed with polyacrylamide gel disc electrophoresis
[lOI * Statistical analysis A split-plot design was employed. The data were evaluated by analysis of variance [ll]. The pooled variation among animals treated alike was used to test the significance of the effect of dietary fat on cholesterol response. The effects of repeated measurements on the same animals across the 15-week period of the experiment and the diet-by-week interaction were tested for significance by the residual error. Three comparisons were made: first, the data of the 2 groups of calves fed the less-saturated fats (SBO + CO) were compared with those of calves fed the more-saturated fats (VS + T); then data from calves fed SBO were compared with those of calves fed CO; finally, similar comparisons were made between the VS- and T-fed calves. Further analyses of pair treatment comparisons were calculated by least significant difference [ 121. Results The calf growth rate, calculated as percentage gain from 0 to 15 weeks, did not vary among dietary groups (Table l), nor were average weekly weights significantly different (Table 2). Plasma cholesterol concentrations increased as the experiment progressed for all 4 groups (P < 0.005). At week 0 plasma cholesterol in mg/dl for SBO, CO, VS and T groups was 90, 91, 108 and 72, respectively; at week 15 the respective values were 161, 141, 113 and 111. Analysis of variance revealed no significant differences in plasma cholesterol concentrations between the 2 unsaturated fats, SBO and CO, nor between the more saturated pair, VS and T. The sum of the plasma cholesterol concentra-
516 TABLE
1
AVERAGE Dietary
BODY group
Soybean Corn
WEIGHT
GAINS Initial
a
oil
oil
Vegetable
shortening
TZdlOW a Five
calves
Final
weight
15-wk
weight
(kg)
(kg)
(%)
43.3
90.5
109
42.6
90.3
112
44.7
94.8
112
37.5
79.1
111
gain
per group.
tion of the groups fed unsaturated fats was significantly different (P < 0.001) from that of the 2 groups fed more saturated fats (Fig. 1 and Table 2). A least significant difference test of all individual pairs showed that both SBO and CO were different from each of the more saturated fats, VS and T (P < 0.01). Statistical analysis of lipoprotein cholesterol revealed that while cholesterol in VLDL, HDL and VHDL was unaffected by diet, cholesterol in LDL was elevated to a greater degree by the SBO and CO diets (Fig. 2 and Table 2) than by the VS and T diets. Cholesterol concentration was greater in the liver (P < 0.001) and fat (P < 0.05) tissue of calves fed SBO and CO than in tissues of calves fed the other diets; muscle concentration was not affected by diet (Table 3). Cholesterol concentration of the liver of the CO group was significantly different than that of the VS and T groups when tested individually by least significant difference (P < 0.01). While the cholesterol concentrations of liver, muscle and fat tissues of the SBO group were not significantly different from those of VS or T
TABLE
2
ANALYSIS
OF
VARIANCE
OF
BLOOD
PLASMA
COMPONENTS
AND
AVERAGE
WEEKLY
WEIGHTS Source
of variation
Plasma
cholesterol
Average
weekly
weight Total
In LDL
In HDL F
df df
F
df
F
F
Treatment (Trt) SBO + CO YS VS+T SBO
vs CO
VSvsT Animals
(within
3
11.27
a
3
1
31.89
a
Weeks
X Trt
Residual
error
1.36
3
1.83
a
1
26.11
0.76
1
0.49
1.34
1
0.08
1.80
1
0.06
1
0.59
1
0.26
1.51
1
(6915)
15d
Weeks
a
1
16
Trt)
8.82
C
12.47
45
d
240
d
2.73
16
(1746)
(103)
23.41
a
9.61
a
15
9
5.73
a
2.27
b
45
48
(133)
(448)
P
in parentheses
conservative
P
df.
are mean i.e.,
weeks
squares
196
3
f
a
d Used
(1675)
e
(443)
for
= 1, weeks
error X Trt
used
to test
= 3, residual
significance. error
= 16.
4.94
16
240
a
0.51 (24)
b
PLASMA
__J
\ 60.
CHOLESTEROL
‘\ :/ 0
,I”
2
4
6
6
IO
12
14
WEEKS Fig. 1. Mean blood plasma cholesterol at weekly intervals (5 calves per group). soybean oil, CO = corn oil, VS = vegetable shortening. and T = tallow.
groups, the mean liver cholesterol concentration calves than in VS or T calves (Table 3).
was slightly
Dietary
groups;
greater
SBO =
in SBO
Discussion The observation that the bovine species is subject to spontaneous atherosclerosis [13-161 and to cholesterol-induced atherogenic changes in the aorta LIPOPROTEIN
CHOLESTEROL
CONCENTRATION
VHDL __-.---------______.-,~.-,~, VLDL
VEG. SHORTENING
I
._.------I------_____
0
___,.,
5
I
VHDL i6vLDL
---_.,.. _______ ‘.j; .__________..VHDL “lo’. ,_
IO
15”mL TIME (WEEKS)
Fig. 2. Mean cholesterol
in various lipoproteins
as mg/dl of blood Plasma (5 cakes
Per group).
518 TABLE
3
MEANS
AND
Dietary
group
Soybean
oil
corn
STANDARD
ERRORS
a
TISSUE
CHOLESTEROL
Liver
oil
Vegetable
OF
b
mg dry
Adipose
1.39
? 0.09
0.30
+ 0.01
0.41
f 0.06
+ 0.11
0.30
+ 0.01
0.37
f 0.04 k 0.02
1.16
?r 0.06
0.29
f 0.01
0.27
1.15
+ 0.07
0.29
? 0.01
0.32
SBO+COvsVS+T
P <
SBO
vs CO
VSvsT a Five
calves
b Biceps
0.001
Nsd
P <
NS
NS
NS
NS
NS
NS
tissue)
tissue
1.55
W
Tall0
shortening
Muscle
@g/100
c
+ 0.05 0.05.
per group.
femoris.
c Perirenal. d NS
= not
significant.
and coronary arteries [17,18] led to the study of effect of feeding saturated and unsaturated fats with and without cholesterol to nonruminating calves [ 191. Calves consuming SBO as 30% of their dietary calories, a level similar to that recommended for humans, have greater plasma cholesterol concentrations than do calves consuming T in the same manner [l-4]. Differences in tissue cholesterol concentrations in liver, fat and muscle were observed, with the SBO-fed calves usually having higher concentrations. Accumulation of tissue cholesterol in response to unsaturated fat feeding has been observed also in growing lambs [20] and rats [21]. In this experiment, there were no cholesterol concentration distribution differences noted between CO- or SBO-fed calves. CO was fed to compare an unsaturated fat that contained by our analysis no detectable linolenic acid with SBO, which contained about 5% linolenic acid (Table 4). Vegetable shortening was fed to ascertain the effect of partial hydrogenation of SBO on the cholesterol distribution in the calves. Although VS contained a lower percentage of saturated fatty acids than T (Table 4), the results from feedings VS were remarkably like those of feeding T. High density lipoproteins constitute about 80% of all adult bovine serum lipoproteins [22,23]; similar observations have been made for the plasma of TABLE
4
LONG
CHAIN
Fatty 14:o
FATTY
acid
ACID Soybean
DISTRIBUTION
IN Corn
oil
DIETARY oil
FATS
a
Vegetable
Tallow
shortening
b
5
16:0
15
15
16
37
12
11 42
16:l
5
18:0
4
1
18:l
25
26
46
18:2
51
58
25
‘18:3
5
a Methyl
esters
&co1
succinate
b Carbon
number:
of
the
1
fatty
column number
acids
were
at 180°C,
and
of double
separated each
bonds.
by
is reported
gas chromatography as a percentage
employing of total
fatty
a 20% acid.
diethylene
519
young calves [24]. At the start of this experiment, two-thirds of the total plasma cholesterol of the calves was found in the HDL fraction. By the end of the experimental period, the situation was reversed for the SBO and CO groups of calves. Two-thirds of the total plasma cholesterol was found in the LDL fraction of these groups; at this point, the plasma cholesterol of the VS and T groups of calves was equally split between the LDL and HDL fractions. When Dryden et al. [25] compared ruminating steers consuming untreated safflower oil with steers consuming safflower oil which was treated to be nonfermentable in the rumen, they found a 3-fold increase in the serum LDL concentration of those consuming the treated oil. At the end of the current experiment with nonruminating calves, a 3-fold increase above initial concentration in LDL cholesterol was observed in SBO and CO groups and a l-fold increase above initial concentration in LDL cholesterol was observed in VS and T groups. At week 0 of our experiment, the average ratio of LDL-C to HDL-C in all calf plasma was 0.46; at week 15, the same ratio was 2.3 for the SBO group, 2.2 for the CO group, 1.5 for the VS group and 1.3 for the T group. Although there has been disagreement among researchers as to the significance of LDL-C/ HDL-C changes, a 1979 Framingham Study perspective [5] states, “A relatively large amount of cholesterol in the lowdensity lipoprotein fraction is atherogenie, whereas that in the high-density fraction appears protective”. Our studies demonstrate clearly that dietary SBO and CO greatly increase LDL-C in the nonruminating calf, the mechanism of which has yet to be explained. Acknowledgements The authors wish to thank Dr. D.F. Cox for guidance in the statistical analyses, E.P. Vining for assistance with the enzymatic cholesterol assays and L.E. Sampson for assistance in the preparation of the manuscript. References 1 Wiggers, K.D., Richard, M.J., Stewart, J.W., Jacobson, N.L. and Berger, P.J., Type and amount of dietary fat affect relative concentration of cholesterol in blood and other tissues of calves, Lipids, 12 (1977) 586. 2 Heeg. T.R.. The Effects of Tallow or Soybean Oil Reconstituted Milk Diets on Plasma and Tissue Cholesterol in Calves, MS. Thesis, Iowa State University Library, Ames, IA, 1977. 3 Barrows, K.K.. Heeg, T.R.. McGilIiard, A.D., Richard, M.J. and Jacobson, N.L., Effect of type of dietary fat on plasma and tissue cholesterol of calves, J. Nutz., 110 (1980) 335. 4 Stewart, J.W., Wiggers. K.D.. Jacobson, N.L. and Berger, P.J.. Effect of various triglycerides on blood and tissue cholesterol of calves, J. Nutr., 108 (1978) 561. 5 Kannel. W.B., Castelli, W.P. and Gordon, T., New perspectives based on the Framingham study, Ann. Intern. Med., 90 (1979) 85. 6 Jacobson, N.L., Bath, D.L., Miller, W.J., Moe, P.W.. Reid, J.T., Schultz, L.H. and Swanson, E.W., Nutrient Requirements of Dairy Cattle, National Academ; of Sciences, National Research Council, Washington, DC, 1978. 7 Technicon Auto-Analyzer Methodology N-24A, Total cholesterol in serum, Technicon Instruments Corp.. Chauncey, NY, 1965. 8 Havel, R.J., Eder. H.A. and Bragdon, J.H., The distribution and chemical composition of ul&racentrifugally separated lipoproteins in human serum, J. Clin. Invest., 34 (1955) 1345. 9 Allain, C.C.. Peon, L.S., Ghan, C.S.G., Richmond, W. and Fu, P.. The enzymatic determination of total serum cholesterol. Clin. Chem.. 20 (1974) 470. 10 Naito, H.K., Wada, M., Ehrhart, L.A. and Lewis, L.A., Polyacrylamide-gel disc-electrophoresis as a screening procedure for serum lipoprotein abnormslities, Clin. Chem.. 19 (1973) 228.
520 11 12 13 14 15 16 17 18 19
20 21 22 23
24 25
Snedecor. G.W. and Cochran, W.G.. Statistical Methods, Iowa State University Press, Ames, IA, 1967. Cochran, W.G. and Cox. G.M.. Experimental Designs, WiIey and Sons, New York, NY 1957. Stehbens, W.E. InthnaI proliferation and spontaneous lipid deposition in the cerebral arteries of sheep and steers, J. AtheroscIer. Res.. 5 (1965) 556. Likar, I.N. and Robinson, R.W., Bovine arterial disease, Part 1 (Localization of lipids in the abdominal aorta in relation to bovine atherosclerosis). Arch. Path., 82 (1966) 555. Skold, B.H.. Jacobson, N.L. and Getty, R., Spontaneous atherosclerosis of bovine, J. Dairy Sci., 50 (1967) 1712. Wiggers, K.D., Jacobson, N.L. and Getty. R., Atherosclerosis in ruminants, J. Anim. Sci.. 32 (1971) 1037. atherosclerosis in the young bovine, Wiggers, K.D.. Jacobson. N.L. and Getty. R., Experimental Atherosclerosis, 14 (1971) 379. Wiggers, K.D.. Jacobson, N.L.. Getty, R. and Richard, M., Mode of cholesterol ingestion and atheros clerosis in the young bovine, Atherosclerosis, 17 (1973) 281. Jacobson, N.L., Richard, M., Berger, P.J. and KIuge. J.P.. Comparative effects of tallow, lard and soybean oil, with and without supplemental cholesterol, on growth, tissue cholesterol and other responses of calves, J. Nutr., 104 (1974) 573. Kirk, R.D.. Elevated cholesterol concentration in muscle of lambs fed unsaturated fats, N.Z. Med. J.. 82 (1975) 58. Angel, A. and Farkas, J., Regulation of cholesterol storage in adipose tissue. J. Lipid Res., 15 (1974) 491. Jonas, A., Physicochemical properties of bovine serum high density lipoprotein, J. Biol. Chem., 247 (1972) 7767. Dryden, F.D., Marcello. J.A., Adams, G.H. and Hale, W.H.. Bovine serum lipids, Part 2 (Lipoprotein quantitative and qualitative composition as influenced by added animal fat diets), J. Anim. Sci., 32 (1971) 1016. Richard, M.J., Unpublished observations (1979). Dryden, F.D., Marcello, J.A.. Cuitun, L.L. and Hale, W.H.. Protein protected fat for ruminants, Part 2 (Serum lipids and lipoproteins), J. Anim. Sci., 40 (1975) 697.