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Effect of dietary linoleic acid on the tissue levels of zinc and copper, and serum high density lipoprotein cholesterol
Atherosclerosis, 50 (1984) 123-132 Elsevier Scientific Publishers Ireland,
123 Ltd.
ATH 03432
Effect of Dietary Linoleic Acid on the Tissue Levels of Zinc and Copper, and Serum High Density Lipoprotein Cholesterol Sung I. Koo * and James S. Ramlet Deportment of Biochemistv,
Oral Roberts University School of Medicine, Tulsa, OK 74171 (U.S.A.) (Received 3 May, 1983) (Revised, received 25 July, 1983) (Accepted 2 August, 1983)
Summary The effects of dietary linoleic acid on the serum level of high density lipoprotein (HDL) cholesterol and its relationship with the tissue status of zinc and copper were examined in adult male rats fed diets differing in the amount of linoleic acid. One group of 9 animals was fed a diet containing hydrogenated coconut oil (4%) and the other was fed a diet containing coconut oil (3.4%) plus linoleic acid (0.6%). Both diets were isocalorically formulated with the equal levels of minerals and other nutrients and contained cholesterol at 1% level. During a 6-week experiment, no differences were observed in food intake and body weight between the two groups. The feeding of linoleic acid produced a significant decrease in serum HDL cholesterol level at 6 weeks and no changes in other lipoproteins and total serum cholesterol and triglyceride. Dietary linoleic acid also significantly lowered the concentrations of zinc in serum and tibia at 6 weeks, while it had no effect on copper contents in these tissues. No changes were observed in the concentration of either zinc or copper in the liver. Linear regression analysis of the 18 pairs of serum zinc and HDL values at 6 weeks indicated a significant positive correlation (r = + 0.65; P < 0.01) between the two parameters, No such relationships were shown between tibia zinc and serum HDL, and between tissue copper and serum HDL. The results indicate that dietary linoleic acid at a relatively low level produces a decrease in serum HDL cholesterol without significantly lowering total serum cholesterol and that the decrease in HDL This work was supported by NIH Heart, Lung and Blood Institute To whom reprint requests should be addressed.
0021-9150/84/$03.00
0 1984 Elsevier Scientific
Publishers
Ireland,
Grant
Ltd.
HL-27531.
124
cholesterol due to linoleic in serum zinc level. Key words:
Copper
-
acid feeding
Dietary
is significantly
linoleic acid
-
High
correlated
density
with the reduction
lipoprotein
-
Serum
cholesterol - Zinc
Introduction The role of certain essential microelements in cholesterol metabolism and its possible link to the development of coronary heart disease (CHD) has been a focus of many recent nutritional studies. Current information concerning the relationship between the body status of essential microelements and etiology of CHD has been reviewed in a recent publication [l]. It is well established that the body status of a microelement is determined not only by the intake but also by the interaction of the element with various dietary ingredients and nutrients at the level of the gastrointestinal tract and during in-vivo metabolism as well [2]. Recently, evidence has been presented that dietary lipids, particularly cholesterol and triglylceride, may significantly influence the nutriture of certain microelements [3,4]. In our recent study [3], we demonstrated that dietary cholesterol, when fed at 1% level, lowered the serum level of zinc with no changes in serum copper, calcium, and magnesium in adult male rats. In their recent study involving young human males, Lukaski et al. [4] suggested that the degree of unsaturation of dietary triglyceride may affect the metabolism of zinc and iron. The data showed that dietary polyunsaturated fat (PUFA) decreased the retention of zinc and iron with no effect on copper, as determined by the dietary intake minus fecal and urinary losses of these microelements. In the light of our earlier observations [3,5] that the serum concentration of zinc is strongly correlated with the serum level of high density lipoprotein (HDL) cholesterol, the present study was undertaken to assess further the zinc-HDL relationship in adult male rats fed triglycerides of different degrees of unsaturation. The present data show that the feeding of PUFA results in decreases in the serum and tibia levels of zinc and that the serum concentration of zinc is positively correlated with the serum level of HDL cholesterol. Materials and Methods Animals and diets
Fischer:344 male rats (Charles River Breeding Lab, Inc., Wilmington, MA) were housed individually in plastic cages with stainless-steel wire bottoms and were fed a commercial rat chow (Ralston Purina Co., St. Louis, MO) ad libitum for about 2 weeks before the initiation of the experiment. The rats with the mean weight of 285 k 5 g were then divided into two treatment groups of 9 rats each and subjected to a light cycle with a 3:00 a.m. to 3:00 p.m. dark period and a 3:00 p.m. to 3:00 a.m. light period throughout the experiment.
125
The two treatment groups consisted of one control group, which was fed a diet containing 4% hydrogenated coconut oil (referred to as SAT) and the other fed 3.4% non-hydrogenated coconut oil plus 0.6% linoleic acid (referred to as UNS). The compositions of the diets were based on the recommendation of the American Institute of Nutrition [6] and are shown in Table 1. The hydrogenated and non-hydrogenated coconut oil used for the SAT and UNS diets contained 0.8 and 2.5% linoleic acid, respectively, and thus the total linoleic acid content of the UNS diet was 0.685%, which provided 1.4% of the total calories of the diet. The polyunsaturated/saturated fat (P/S) ratios of the SAT and UNS diets were 0.01 and 0.18, respectively. The two groups of rats were fed ad libitum and allowed free access to deionized water delivered by a continuous-flow automatic watering system. The concentrations of zinc and copper in deionized water given were below the detection limits for the elements (0.002 pg/ml) by the flame atomic absorption spectrophotometer used. TABLE
1
THE COMPOSITION
OF EXPERIMENTAL
Inaredients
Casein Corn starch Sucrose Mineral mix (AIN) a Vitamin mix (AIN) a DL-Methionine Choline-Cl (50%) Cellulose (Solka Floe) Cholesterol Coconut oil, hydrogenated Coconut oil, edible b Linoleic acid
DIETS Percentage
b
of diet
SAT diet
UNS diet
20.0 15.0 50.0 3.5 1.0 0.3 0.2 5.0 1.0 4.0
20.0 15.0 50.0 3.5 1.0 0.3 0.2 5.0 1.0 3.4 0.6
a American Institute of Nutrition formula (ref. [6]). b Coconut oil, hydrogenated and edible (non-hydrogenated), PA. The typical fatty acid compositions of hydrogenated follows (% weight). Fatty acid
Non-hydrogenated
Octanoic Decanoic Laurie Myristic Palmitic Hexadecenoic Stearic Oleic Linoleic
7.6 1.3 48.2 16.6 8.0 1.0 3.8 5.0 2.5
were purchased from Dyets Inc., Bethlehem, and non-hydrogenated coconut oil were as
,
Hydrogenated 7.6 1.3 48.2 16.6 8.7 0.3 9.0 1.5 0.8
126
Experimental procedure At 2 and 6 weeks of dietary treatment, blood samples (approximately 1.0 ml) were taken via the orbital sinus [7] under slight anesthesia with diethylether during the mid-dark phase (8:30 a.m. to 9:30 a.m.) of the light cycle after an 18-h fast. Serum was separated by centrifuging at 1000 X g for 30 min at 4°C and used within 48 h for the fractionation of lipoprotein classes by a slight modification of Bronzert and Brewer’s procedure [8]. The very low density lipoprotein (VLDL) fraction was separated from low density (LDL) and high density lipoproteins (HDL) by using a Beckman Airfuge with an A-100 fixed angle rotor (Beckman Instruments, Inc., Palo Alto, CA). The HDL fraction was separated by precipitation with Mg*‘/ phosphotungstic acid [9]. The concentrations of cholesterol and triglyceride were determined by enzymatic methods described earlier [lO,ll]. The average recovery of total serum cholesterol after the fractionation procedure was 99.0 + 1.4%. Tissue and serum concentrations of zinc and copper were determined by using an atomic absorption spectrophotometer equipped with a double-beam background corrector and microprocessor. Serum samples were diluted 1: 3 with deionized-distilled H,O prior to analysis. Liver and left tibia were removed from each rat killed by cervical dislocation after anesthesia. The whole liver was weighed, lyophilized, and pulverized. A portion (approximately 1 .O g) of the pulverized liver and the whole tibia were charred in concentrated HNO, and wet-ashed in a 2 : 1 (v/v) mixture of HNO, and 70% HClO, on a hot plate. After appropriate dilutions of the wet-ashed samples, the concentrations of liver zinc and copper, and tibia zinc were determined by a flame atomic absorption spectrophotometry (Perkin-Elmer Co., Norwalk, CT). The copper content of tibia was determined by using a flameless atomic absorption spectrometer (HGA-500 Perkin-Elmer Co., Norwalk, CT) equipped with an automatic sampler. For tibia copper analysis, the wet-ashed sample (20 ~1) was charred at 800°C for 20 s and atomized at 2650°C. The solutions of metal standards were made from the reference standards (Fisher Scientific Co., Fair Lawn, NJ). The wavelengths for zinc and copper analysis were set at 213.9 and 324.7 nm, respectively. Data were analyzed statistically by two-way analysis of variance when the effects of diets and time were analyzed, and t-test, when only the effects of diets were compared. Results Throughout the study, no difference in either diet consumption or body weight was noted between the two groups of animals fed saturated (SAT) and unsaturated (UNS) fats (Table 2). In addition, careful examination of the rats fed the SAT diet failed to detect any visible signs of essential fatty acid (EFA) deficiency such as rough hair and dermal lesions on the tail and feet, although it does not necessarily indicate the absence of early biochemical lesions, which might have been induced by essential fatty acid depletion. The feeding of UNS diet produced a significant (P < 0.05) decrease in zinc at 6 weeks, compared to the diet (containing hydrogenated coconut oil (SAT diet),
127 TABLE
2
EFFECT OF EXPERIMENTAL THE RATS
DIETS ON FOOD
Week after dietary Initial Diet consumption SAT UNS
CONSUMPTION
a AND
BODY WEIGHT
a OF
treatment
2nd wk
4th wk
6th wk
(g/rat/day) 26.9+1.0’ 25.4+0.5 a
Body weight (g/rat) SAT UNS
284 285
a Mean k SEM of 9 rats per group. significantly different (P > 0.05).
*5” k5”
25.5 f 1.0 a 24.5 rtO.7 a
368 360
f8s k8”
Values in the same column
26.8* 24.7*
444 428 sharing
1.4s 1.0”
26.8+ 25.0+
*14” fl0’ a common
470 453
1.2’ a 1.0 a
*15a +11s
superscript
are not
although such an effect was not evident at 2 weeks (Table 3). There were no differences (P > 0.05)in serum copper levels at either 2 or 6 weeks of dietary treatment. To further evaluate the zinc and copper status of the animals, the concentrations of the minerals in the liver and tibia were determined at the end of the 6-week experiment (Table 4). The feeding of linoleic acid did not affect the concentrations of zinc and copper in the liver but resulted in a significant (P < 0.01) decrease in the zinc concentration of the tibia with no effect on the level of copper, compared with the group fed SAT diet (Table 4). The serum concentrations of total triglyceride and cholesterol were not significantly (P > 0.05) affected by dietary linoleic acid during the 6-week period (Table 5). Also, there were no changes in the levels of VLDL- and LDL cholesterol, as determined at both 2 and 6 weeks. The level of HDL cholesterol did not significantly (P > 0.05) change at 2 weeks of dietary treatment, although it tended to be lowered by linoleic acid feeding. A significant decrease in HDL cholesterol was, however, clearly noted at 6 weeks in the rats fed linoleic acid. TABLE
3
EFFECT OF EXPERIMENTAL DIETS ON SERUM LEVELS= OF ZINC DETERMINED AT 2nd AND 6th WEEKS OF DIETARY TREATMENT Dietary treatment
SAT UNS a MeankSEM significantly
Week after dietary
COPPER,
AS
treatment 6th wk
2nd wk Zn
cu
(pg/mI)
@g/ml)
1.40*0.03 1.37kO.02
AND
a ’
1.21 f0.04 1.11 f0.05
of 9 rats per group. different (P -c 0.05).
ZN/Cu
a a
1.16+0.04” 1.26+0.06’
Zn
cu
(pg/mI)
@g/ml)
1.52*0.04’ 1.40*0.03 b
1.31 f 0.05 a 1.28 + 0.05 ’
Values in the same column
not sharing
Zn/Cu
a common
1.17*0.03 1.12+0.05 superscript
B ’ are
128 TABLE
4
EFFECTS OF EXPERIMENTAL IN THE LIVER AND TIBIA
DIETS ON THE CONCENTRATIONS
Total weight
a OF ZINC AND COPPER
Copper (gg/g fresh tissue)
(9)
Zinc (pg/g
Liver SAT UNS
14.9 f 0.9 a 14.8 f 0.8 ’
25.78*1.98’ 25.22zt1.42’
5.39 f 0.43 a 5.26 + 0.28 ’
Tibia SAT UNS
0.67 f 0.03 a 0.69 + 0.02 ’
155.78 f 3.01 ’ 145.22 f 2.27 b
0.33 + 0.09 B 0.20 * 0.04 a
Tissue
’ Mean f SEM of 9 rats per group. significantly different (P -C 0.01).
fresh tissue)
Values in the same column
not sharing
a common
superscript
are
In view of our earlier findings of a close correlation between the zinc status and HDL cholesterol [3,5], the relationship between the two parameters was further examined in the present study. Linear regression analyses, based on the 18 pairs of either serum or tissue level of zinc and serum HDL cholesterol as obtained at 6 weeks, showed that the serum level of zinc was positively correlated (r = +0.65; P < 0.01) with serum HDL cholesterol (Fig. 1). No correlation (P > 0.05) was noted TABLE
5
EFFECTS OF EXPERIMENTS LIPOPROTEIN CHOLESTEROL
DIETS
ON THE
Serum lipids
Weeks after dietary 2nd (mg/lOO
SERUM
LEVELSa
OF TOTAL
LIPIDS
AND
treatment
ml)
6th (mg/lOO
Total triglyceride SAT UNS
61.0 f 4.3 a 55.7k4.2 a
84.9k33.7 b 84.9 f 8.9 b
Total cholesterol SAT UNS
99.3 f 9.7 a 97.1 f 9.4 B
110.1 f 9.7 a 91.3*7.4a
VLDL cholesterol SAT UNS
25.8 f 3.3 ’ 24.3 f 2.7 ’
32.2 f 3.0 ’ 27.3 f 4.2 a
LDL cholesterol SAT UNS
15.8 f 2.3 ’ 26.6 f 5.7 ’
18.8 f 1.6 a 17.8k2.1 ’
53.8 k5.5 a.b 46.2 f 3.7 b
59.1 f 6.4 ’ 46.2 f 3.2 b
ml)
HDL cholesterol SAT UNS
a Mean f SEM of 9 rats per group. Values in the same column common superscript are significantly different (P i 0.05).
or row for each parameter
not sharing
a
129
between the levels of tibia (or liver) zinc and HDL cholesterol. No relationships (P > 0.05) were indicated between serum (or tissue) concentrations of copper and any of the major lipoprotein fractions. Discussion
The hypocholesterolemic effect of dietary polyunsaturated fatty acid (PUFA) has been well-documented in animals as well as in humans (e.g. [12] and [13]). Such an effect of PUFA has been considered as beneficial with regard to reducing risk of CHD, although it still remains to be established whether the lowering of serum cholesterol levels by dietary means in general reduces the incidence of CHD [14]. In recent years, much attention has been directed toward the distribution of serum cholesterol among the major lipoprotein fractions as affected by dietary PUFA. Conflicting results have been reported as to whether the dietary PUFA-induced hypocholesterolemia is due to a decrease in VLDL- LDL-, and/or HDL cholesterol [12,13,15-191. The present data showed that the feeding of 0.6% linoleic acid for 6 weeks produced a significant decrease (22%) in serum HDL cholesterol, compared with a diet containing hydrogenated coconut oil with no supplemental linoleic acid. Under the present experimental conditions, dietary linoleic acid did not lower significantly the serum level of total cholesterol or the serum concentrations of VLDL- and LDL cholesterol. The levels of total serum cholesterol, however, tended to be lower in the group fed UNS diet at both 2 and 6 weeks, compared to the values for the rats fed SAT diet. It should also be pointed out that there appeared to be a transient rise in LDL cholesterol at 2 weeks ip the rats fed UNS diet (Table 5), although the increase was not statistically significant (P > 0.05). Such a response to dietary PUFA during an early phase of dietary treatment has been reported to occur, when the diet contains both cholesterol and unsaturated fat [30]. Previously, Durrington et al. [19] reported that, in healthy normolipidemic men, a high PUFA diet (P/S = 2.82) with a low cholesterol intake (112 mg/day) decreased the serum levels of total cholesterol by 18.7% compared with a low PUFA diet (P/S = 0.23) with a high cholesterol intake (667 mg/day), and that the decrease in total serum cholesterol was due primarily to a decrease in LDL cholesterol. In a similar study with normolipidemic subjects, Tan et al. [18] showed that, when compared to a high-saturated fat diet with a daily cholesterol intake of 1021 mg (P/S = 0.4), a high-PUFA, low-cholesterol (98 mg/day) diet decreased total serum cholesterol by lowering the cholesterol content in VLDL (59%) LDL (15%), and HDL (30%). In their recent series of human studies using a constant level of cholesterol (400 mg/day), Shepherd et al. showed that a high-PUFA diet (P/S = 4.0) lowered total plasma cholesterol by 23-24%, compared with a saturated fat diet (P/S = 0.25) by decreasing the cholesterol content in VLDL (25-27%), LDL (20-30%), and HDL (20-33%). In other recent animal [12] and human [16] studies, similar effects of dietary PUFA on all major lipoprotein fractions have been reported. In the present study, the dietary level of cholesterol was kept at 1% for both diets. The lowering of HDL cholesterol was observed before the hypocholesterolemic
130
effect of PUFA became clearly evident. Although the present results do not provide information concerning diet-induced changes in fatty acid composition of lipoproteins, previous studies [12,13,15,19] have demonstrated that the feeding of polyunsaturated fat increases the content of linoleic acid (C,sZ2) of lipoprotein lipids and decreases that of oleic acid (C,,: i) and palmitic acid (C,,:,). The degree of such changes is directly related to the extent of modification of the chemical and physical properties and hence metabolism (or clearance) rate of serum lipoproteins. The reason for the rather selective effect of PUFA on HDL cholesterol observed in the present experiment might be that the UNS diet contained a relatively small amount of linoleic acid with a low P/S ratio (0.18). Also, it might be explained by the general lack of response of the rat to diet-induced hypercholesterolemia. From the aforementioned and present studies, it is evident that the effects of dietary PUFA on serum lipoprotein levels depend upon the hypocholesterolemia induced and the concentration of PUFA relative to that of other dietary lipids, particularly, saturated fat and cholesterol. Based upon the above and present observations, the non-specific nature of PUFA effect in altering the levels of serum lipoproteins should be carefully interpreted. It is yet to be known whether the demonstrated effects of PUFA on the levels of total serum cholesterol and lipoproteins are beneficial in reducing risk of CHD. In the light of the suggested role of VLDL and LDL in atherogenesis [20,21] and well-documented inverse relationship between serum HDL level and incidence of CHD [22-281, it should be pointed out that the potential benefits of the hypocholesterolemic and LDL-lowering effects of PUFA must be evaluated against the suggested detrimental effect of a decrease in HDL produced by dietary PUFA [13,16,18]. In conjunction with the HDL-lowering effect of dietary PUFA, it is of particular interest to note that, in the present study, the serum level of zinc in the linoleic acid-fed rats were significantly lower at 6 weeks, relative to the level of zinc in the rats fed saturated fat. The data also show that the serum concentration of HDL cholesterol was significantly correlated (r = + 0.65; P -c0.01) with serum zinc status (Fig. 1). The phenomenon of positive correlation between serum zinc and HDL cholesterol has been first demonstrated in our previous studies using adult male rats depleted of zinc [5] and those fed cholesterol [3]. In the first study [5], we have shown that an acute depletion of zinc produces a selective decline in HDL cholesterol and that the serum status of zinc is strongly correlated (r = +0.81; P < 0.01) with serum HDL
Y -89.80 + 97.73.x r = + 0.65 PC 0.01
0: SAT 0: UNS
Serum zinc, ppm
1. Relationship
between
the serum levels of zinc and HDL cholesterol.
131
cholesterol. Subsequently [3], we have also observed that dietary cholesterol at 1% level significantly decreases the serum levels of both ‘zinc and HDL cholesterol and At that the two parameters are significantly correlated (r = + 0.57; P < 0.001). present, it is not known whether the zinc-lowering event mediated by dietary linoleic acid or dietary cholesterol is linked in a specific manner to the metabolism (e.g., synthesis and/or secretion) of HDL. In our previous studies [5,29] using zinc-depleted rats, we have postulated that the nutritional status of zinc may affect the synthesis of the apolipoproteins of chylomicrons and possibly of HDL. Based on the present findings, it is difficult to determine whether the rats fed linoleic acid were marginally deficient in zinc, although the levels of zinc in serum and tibia of the rats were significantly lowered by dietary linoleic acid. In the present study, the diets used were adequate in zinc (30 ppm), and the serum and tissue levels of zinc in these rats were within the ‘normal’ range. In addition, the food intake and body growth of the animals were not affected by the feeding of linoleic acid. However, the possibility that dietary linoleic acid may adversely affect the nutritional status of zinc has been suggested in a recent study using human subjects [4]. The study showed that, with the average zinc intake of 25 mg/day, the retentions of zinc effected by saturated and polyunsaturated fats were 5.9 and 0.6 mg/day, respectively, when the retention was determined by the intake minus fecal and urinary losses. This suggests that the ingestion of PUFA may influence the metabolism of zinc, perhaps interfering with the intestinal absorption and/or the turnover of zinc. The observations thus far made indicate that, for future studies on this subject, careful consideration should be given to the amounts and types of dietary lipids and their effects on the metabolism and body status of zinc. Further studies are underway to define the interactive effects of dietary triglyceride, cholesterol and zinc on the metabolism of HDL and zinc. In summary, the present study has shown that dietary PUFA (linoleic acid) at a relatively low level produces a decrease in serum HDL cholesterol without significantly lowering total serum cholesterol and that the decrease in HDL-cholesterol due to PUFA feeding is associated with the reduction in serum zinc. The practical significance of the zinc- and HDL-lowering effects of dietary PUFA remains to be evaluated. In addition, the phenomenon of the positive relationship between serum zinc and HDL cholesterol, as repeatedly observed in the present and previous studies [3,5], warrants further investigation into the possible biochemical role(s) of zinc in HDL metabolism. References 1 Mertz, W., Trace minerals and atherosclerosis, Fed. Proc., 41 (1982) 2807. 2 Underwood, E.J., I. Zinc in animal tissues and fluids. In: Trace Elements in Human and Animal Nutrition, 4th edition, Academic Press, New York, NY, 1977, pp. 196-205. 3 Koo, S.I. and Ramlet, J.S., Dietary cholesterol decreases the serum level of zinc - Further evidence for the positive relationship between serum zinc and high-density lipoproteins, Amer. J. Clin. Nutr., 37 (1983) 918. 4 Lukaski, H.C., Klevay, L.M., Bolonchuk, W.W. et al., Influence of dietary lipids on iron, zinc, and copper retention in trained athletes, Fed. Proc., 41 (1982) 275. 5 Koo, S.I. and Williams, D.A., Relationship between the nutritional status of zinc and cholesterol concentration of serum lipoproteins in adult male rats, Amer. J. Clin. Nutr., 34 (1981) 2376.
132
Institute of Nutrition, Report of the American Institute of Nutrition Ad Hoc 6 The American Committee. on standards for nutritional studies, J. Nutr., 107 (1977) 1340. of orbital bleeding technic to rapid serial blood studies, Proc. Sot. Exp. Biol. 7 Riley, V., Adaptation Med., 104 (1960) 751. for measuring cholesterol in plasma lipopro8 Bronzert, T.J. and Brewer, Jr., H.B., New micromethod tein fractions, Clin. Chem., 23 (1977) 2089. M.F., Stone, P., Ellis, S. and Colwell, J.A., Cholesterol determination in high-density 9 Lopes-Virella, lipoproteins separated by three different methods, Clin. Chem., 23 (1977) 882. of total 10 Allain, C.C., Poon, L.S., Chan, C.S.G., Richmond, W. and Fu, P.C., Enzymatic determination serum cholesterol, Clin. Chem., 20 (1974) 470. determination of serum triglylcerides by the use of enzymes, 11 Bucolo, G. and David, H., Quantitative Clin. Chem., 19 (1973) 476. 12 Nicolosi, R.J., Marlett, J.A., Morello, A.M., Flanagan, S.A. and Hegsted, D.M., Influence of dietary unsaturated and saturated fat on the plasma lipoproteins of mongolian gerbils, Atherosclerosis, 38 (1980) 359. 13 Shepherd, J., Packard, C.J., Patsch, J.R., Gotto, Jr., A.M. and Taunton, O.D., Effects of dietary polyunsaturated and saturated fat on the properties of high density lipoproteins and the metabolism of apolipoprotein A-l, J. Clin. Invest., 61 (1978) 1582. 14 Food and Nutrition Board, National Research Council, Cardiovascular disease. In: Toward Healthful Diets, National Academy of Sciences, Washington, DC, 1980, pp. 8-12. 15 Shepherd, J., Packard, C.J., Grundy, SM. et al., Effects of saturated and polyunsaturated fat diets on the chemical composition and metabolism of low density lipoproteins in man, J. Lipid Res., 21 (1980) 91. 16 Vessby, B., Boberg, J., Gustafsson, I-B. et al., Reduction of high density lipoprotein cholesterol and apolipoprotein A-l concentrations by a lipid-lowering diet, Atherosclerosis, 35 (1980) 21. 17 Jackson, R.L., Taunton, O.D., Morrisett, J.D. and Gotto, Jr., A.M., The role of dietary polyunsaturated fat, Cir. Res., 42 (1978) 447. 18 Tan, M.H., Dickinson, M.A. and Albers, J.J., The effect of a high cholesterol and saturated fat diet on serum high-density lipoprotein-cholesterol, apoprotein A-l, and apoprotein E levels in normolipidemic humans, Amer. J. Clin. Nutr., 33 (1980) 2557. 19 Durrington, P.N., Bolton, C.H., Hartog, M., Angelinetta, R., Emmett, P. and Furniss, S., The effect of a low-cholesterol, high-polyunsaturate diet on serum lipid levels, apolipoprotein B levels and triglyceride fatty acid composition, Atherosclerosis, 27 (1977) 465. 20 Kannel, W.B., Castelli, W.P., Gordon, T. and McNamara, P.M., Serum cholesterol, lipoproteins, and the risk of coronary heart disease, Ann. Int. Med., 74 (1971) 1. 21 Camejo, G., Interaction of low density lipoproteins with arterial constituents - Its relationship with atherogenesis. In: C.E. Day and R.S. Levy (Eds.), Low Density Lipoproteins, Plenum Press, New York, NY, 1976, p. 351. 22 Carlson, L.A. and Ericsson, M., Quantitative and qualitative serum lipoprotein analysis, Part 2 (Studies in male survivors of myocardial infarction), Atherosclerosis, 21 (1975) 435. 23 Miller, N.E., Thelle, D.S., Ferde, O.H. and Mjos, O.D., The Tromso Heart Study, Lancet, i (1977) 965. 24 Rhoads, G.G., Gulbrandsen, C.L. and Kagan, A., Serum lipoprotein and coronary heart disease in a population study of Hawaii Japanese men, N. Engl. J. Med., 294 (1976) 293. 25 Castelli, W.P., Doyle, J.T., Gorden, T. et al., HDL-cholesterol and other lipids in coronary heart disease - The Cooperative Lipoprotein Phenotyping Study, Circulation, 55 (1977) 767. 26 Gordon, T., Castelli, W.P., Hjortland, M.J., Kannel, W.B. and Dawber, T.R., High density lipoprotein as a protective factor against coronary heart disease, Amer. J. Med., 62 (1977) 707. 27 Tyroler, H.A., Hames, C.G., Krishen, I., Hayden, S., Cooper, G. and Cassel, J.C., Black-white differences in serum lipids and lipoproteins in Evans County, Prevent. Med., 4 (1975) 541. 28 Miller, G.J. and Miller, N.E., Plasma-high-density-lipoprotein concentration and development of ischaemic heart-disease, Lancet, i (1975) 16. 29 Koo, S.I. and Turk, D.E., Effect of zinc deficiency on intestinal transport of triglycerides in the rat, J. Nutr., 107 (1977) 909. 30 Reiser, R., Clark, D.A., Sorrels, M.F., Gibson, B.S., Williams, M.C. and Wilson, F.H., Tissue cholesterol transport as modified by diet cholesterol and the nature of diet fat, J. Atheroscler. Res., 6 (1966) 565.
123 Ltd.
ATH 03432
Effect of Dietary Linoleic Acid on the Tissue Levels of Zinc and Copper, and Serum High Density Lipoprotein Cholesterol Sung I. Koo * and James S. Ramlet Deportment of Biochemistv,
Oral Roberts University School of Medicine, Tulsa, OK 74171 (U.S.A.) (Received 3 May, 1983) (Revised, received 25 July, 1983) (Accepted 2 August, 1983)
Summary The effects of dietary linoleic acid on the serum level of high density lipoprotein (HDL) cholesterol and its relationship with the tissue status of zinc and copper were examined in adult male rats fed diets differing in the amount of linoleic acid. One group of 9 animals was fed a diet containing hydrogenated coconut oil (4%) and the other was fed a diet containing coconut oil (3.4%) plus linoleic acid (0.6%). Both diets were isocalorically formulated with the equal levels of minerals and other nutrients and contained cholesterol at 1% level. During a 6-week experiment, no differences were observed in food intake and body weight between the two groups. The feeding of linoleic acid produced a significant decrease in serum HDL cholesterol level at 6 weeks and no changes in other lipoproteins and total serum cholesterol and triglyceride. Dietary linoleic acid also significantly lowered the concentrations of zinc in serum and tibia at 6 weeks, while it had no effect on copper contents in these tissues. No changes were observed in the concentration of either zinc or copper in the liver. Linear regression analysis of the 18 pairs of serum zinc and HDL values at 6 weeks indicated a significant positive correlation (r = + 0.65; P < 0.01) between the two parameters, No such relationships were shown between tibia zinc and serum HDL, and between tissue copper and serum HDL. The results indicate that dietary linoleic acid at a relatively low level produces a decrease in serum HDL cholesterol without significantly lowering total serum cholesterol and that the decrease in HDL This work was supported by NIH Heart, Lung and Blood Institute To whom reprint requests should be addressed.
0021-9150/84/$03.00
0 1984 Elsevier Scientific
Publishers
Ireland,
Grant
Ltd.
HL-27531.
124
cholesterol due to linoleic in serum zinc level. Key words:
Copper
-
acid feeding
Dietary
is significantly
linoleic acid
-
High
correlated
density
with the reduction
lipoprotein
-
Serum
cholesterol - Zinc
Introduction The role of certain essential microelements in cholesterol metabolism and its possible link to the development of coronary heart disease (CHD) has been a focus of many recent nutritional studies. Current information concerning the relationship between the body status of essential microelements and etiology of CHD has been reviewed in a recent publication [l]. It is well established that the body status of a microelement is determined not only by the intake but also by the interaction of the element with various dietary ingredients and nutrients at the level of the gastrointestinal tract and during in-vivo metabolism as well [2]. Recently, evidence has been presented that dietary lipids, particularly cholesterol and triglylceride, may significantly influence the nutriture of certain microelements [3,4]. In our recent study [3], we demonstrated that dietary cholesterol, when fed at 1% level, lowered the serum level of zinc with no changes in serum copper, calcium, and magnesium in adult male rats. In their recent study involving young human males, Lukaski et al. [4] suggested that the degree of unsaturation of dietary triglyceride may affect the metabolism of zinc and iron. The data showed that dietary polyunsaturated fat (PUFA) decreased the retention of zinc and iron with no effect on copper, as determined by the dietary intake minus fecal and urinary losses of these microelements. In the light of our earlier observations [3,5] that the serum concentration of zinc is strongly correlated with the serum level of high density lipoprotein (HDL) cholesterol, the present study was undertaken to assess further the zinc-HDL relationship in adult male rats fed triglycerides of different degrees of unsaturation. The present data show that the feeding of PUFA results in decreases in the serum and tibia levels of zinc and that the serum concentration of zinc is positively correlated with the serum level of HDL cholesterol. Materials and Methods Animals and diets
Fischer:344 male rats (Charles River Breeding Lab, Inc., Wilmington, MA) were housed individually in plastic cages with stainless-steel wire bottoms and were fed a commercial rat chow (Ralston Purina Co., St. Louis, MO) ad libitum for about 2 weeks before the initiation of the experiment. The rats with the mean weight of 285 k 5 g were then divided into two treatment groups of 9 rats each and subjected to a light cycle with a 3:00 a.m. to 3:00 p.m. dark period and a 3:00 p.m. to 3:00 a.m. light period throughout the experiment.
125
The two treatment groups consisted of one control group, which was fed a diet containing 4% hydrogenated coconut oil (referred to as SAT) and the other fed 3.4% non-hydrogenated coconut oil plus 0.6% linoleic acid (referred to as UNS). The compositions of the diets were based on the recommendation of the American Institute of Nutrition [6] and are shown in Table 1. The hydrogenated and non-hydrogenated coconut oil used for the SAT and UNS diets contained 0.8 and 2.5% linoleic acid, respectively, and thus the total linoleic acid content of the UNS diet was 0.685%, which provided 1.4% of the total calories of the diet. The polyunsaturated/saturated fat (P/S) ratios of the SAT and UNS diets were 0.01 and 0.18, respectively. The two groups of rats were fed ad libitum and allowed free access to deionized water delivered by a continuous-flow automatic watering system. The concentrations of zinc and copper in deionized water given were below the detection limits for the elements (0.002 pg/ml) by the flame atomic absorption spectrophotometer used. TABLE
1
THE COMPOSITION
OF EXPERIMENTAL
Inaredients
Casein Corn starch Sucrose Mineral mix (AIN) a Vitamin mix (AIN) a DL-Methionine Choline-Cl (50%) Cellulose (Solka Floe) Cholesterol Coconut oil, hydrogenated Coconut oil, edible b Linoleic acid
DIETS Percentage
b
of diet
SAT diet
UNS diet
20.0 15.0 50.0 3.5 1.0 0.3 0.2 5.0 1.0 4.0
20.0 15.0 50.0 3.5 1.0 0.3 0.2 5.0 1.0 3.4 0.6
a American Institute of Nutrition formula (ref. [6]). b Coconut oil, hydrogenated and edible (non-hydrogenated), PA. The typical fatty acid compositions of hydrogenated follows (% weight). Fatty acid
Non-hydrogenated
Octanoic Decanoic Laurie Myristic Palmitic Hexadecenoic Stearic Oleic Linoleic
7.6 1.3 48.2 16.6 8.0 1.0 3.8 5.0 2.5
were purchased from Dyets Inc., Bethlehem, and non-hydrogenated coconut oil were as
,
Hydrogenated 7.6 1.3 48.2 16.6 8.7 0.3 9.0 1.5 0.8
126
Experimental procedure At 2 and 6 weeks of dietary treatment, blood samples (approximately 1.0 ml) were taken via the orbital sinus [7] under slight anesthesia with diethylether during the mid-dark phase (8:30 a.m. to 9:30 a.m.) of the light cycle after an 18-h fast. Serum was separated by centrifuging at 1000 X g for 30 min at 4°C and used within 48 h for the fractionation of lipoprotein classes by a slight modification of Bronzert and Brewer’s procedure [8]. The very low density lipoprotein (VLDL) fraction was separated from low density (LDL) and high density lipoproteins (HDL) by using a Beckman Airfuge with an A-100 fixed angle rotor (Beckman Instruments, Inc., Palo Alto, CA). The HDL fraction was separated by precipitation with Mg*‘/ phosphotungstic acid [9]. The concentrations of cholesterol and triglyceride were determined by enzymatic methods described earlier [lO,ll]. The average recovery of total serum cholesterol after the fractionation procedure was 99.0 + 1.4%. Tissue and serum concentrations of zinc and copper were determined by using an atomic absorption spectrophotometer equipped with a double-beam background corrector and microprocessor. Serum samples were diluted 1: 3 with deionized-distilled H,O prior to analysis. Liver and left tibia were removed from each rat killed by cervical dislocation after anesthesia. The whole liver was weighed, lyophilized, and pulverized. A portion (approximately 1 .O g) of the pulverized liver and the whole tibia were charred in concentrated HNO, and wet-ashed in a 2 : 1 (v/v) mixture of HNO, and 70% HClO, on a hot plate. After appropriate dilutions of the wet-ashed samples, the concentrations of liver zinc and copper, and tibia zinc were determined by a flame atomic absorption spectrophotometry (Perkin-Elmer Co., Norwalk, CT). The copper content of tibia was determined by using a flameless atomic absorption spectrometer (HGA-500 Perkin-Elmer Co., Norwalk, CT) equipped with an automatic sampler. For tibia copper analysis, the wet-ashed sample (20 ~1) was charred at 800°C for 20 s and atomized at 2650°C. The solutions of metal standards were made from the reference standards (Fisher Scientific Co., Fair Lawn, NJ). The wavelengths for zinc and copper analysis were set at 213.9 and 324.7 nm, respectively. Data were analyzed statistically by two-way analysis of variance when the effects of diets and time were analyzed, and t-test, when only the effects of diets were compared. Results Throughout the study, no difference in either diet consumption or body weight was noted between the two groups of animals fed saturated (SAT) and unsaturated (UNS) fats (Table 2). In addition, careful examination of the rats fed the SAT diet failed to detect any visible signs of essential fatty acid (EFA) deficiency such as rough hair and dermal lesions on the tail and feet, although it does not necessarily indicate the absence of early biochemical lesions, which might have been induced by essential fatty acid depletion. The feeding of UNS diet produced a significant (P < 0.05) decrease in zinc at 6 weeks, compared to the diet (containing hydrogenated coconut oil (SAT diet),
127 TABLE
2
EFFECT OF EXPERIMENTAL THE RATS
DIETS ON FOOD
Week after dietary Initial Diet consumption SAT UNS
CONSUMPTION
a AND
BODY WEIGHT
a OF
treatment
2nd wk
4th wk
6th wk
(g/rat/day) 26.9+1.0’ 25.4+0.5 a
Body weight (g/rat) SAT UNS
284 285
a Mean k SEM of 9 rats per group. significantly different (P > 0.05).
*5” k5”
25.5 f 1.0 a 24.5 rtO.7 a
368 360
f8s k8”
Values in the same column
26.8* 24.7*
444 428 sharing
1.4s 1.0”
26.8+ 25.0+
*14” fl0’ a common
470 453
1.2’ a 1.0 a
*15a +11s
superscript
are not
although such an effect was not evident at 2 weeks (Table 3). There were no differences (P > 0.05)in serum copper levels at either 2 or 6 weeks of dietary treatment. To further evaluate the zinc and copper status of the animals, the concentrations of the minerals in the liver and tibia were determined at the end of the 6-week experiment (Table 4). The feeding of linoleic acid did not affect the concentrations of zinc and copper in the liver but resulted in a significant (P < 0.01) decrease in the zinc concentration of the tibia with no effect on the level of copper, compared with the group fed SAT diet (Table 4). The serum concentrations of total triglyceride and cholesterol were not significantly (P > 0.05) affected by dietary linoleic acid during the 6-week period (Table 5). Also, there were no changes in the levels of VLDL- and LDL cholesterol, as determined at both 2 and 6 weeks. The level of HDL cholesterol did not significantly (P > 0.05) change at 2 weeks of dietary treatment, although it tended to be lowered by linoleic acid feeding. A significant decrease in HDL cholesterol was, however, clearly noted at 6 weeks in the rats fed linoleic acid. TABLE
3
EFFECT OF EXPERIMENTAL DIETS ON SERUM LEVELS= OF ZINC DETERMINED AT 2nd AND 6th WEEKS OF DIETARY TREATMENT Dietary treatment
SAT UNS a MeankSEM significantly
Week after dietary
COPPER,
AS
treatment 6th wk
2nd wk Zn
cu
(pg/mI)
@g/ml)
1.40*0.03 1.37kO.02
AND
a ’
1.21 f0.04 1.11 f0.05
of 9 rats per group. different (P -c 0.05).
ZN/Cu
a a
1.16+0.04” 1.26+0.06’
Zn
cu
(pg/mI)
@g/ml)
1.52*0.04’ 1.40*0.03 b
1.31 f 0.05 a 1.28 + 0.05 ’
Values in the same column
not sharing
Zn/Cu
a common
1.17*0.03 1.12+0.05 superscript
B ’ are
128 TABLE
4
EFFECTS OF EXPERIMENTAL IN THE LIVER AND TIBIA
DIETS ON THE CONCENTRATIONS
Total weight
a OF ZINC AND COPPER
Copper (gg/g fresh tissue)
(9)
Zinc (pg/g
Liver SAT UNS
14.9 f 0.9 a 14.8 f 0.8 ’
25.78*1.98’ 25.22zt1.42’
5.39 f 0.43 a 5.26 + 0.28 ’
Tibia SAT UNS
0.67 f 0.03 a 0.69 + 0.02 ’
155.78 f 3.01 ’ 145.22 f 2.27 b
0.33 + 0.09 B 0.20 * 0.04 a
Tissue
’ Mean f SEM of 9 rats per group. significantly different (P -C 0.01).
fresh tissue)
Values in the same column
not sharing
a common
superscript
are
In view of our earlier findings of a close correlation between the zinc status and HDL cholesterol [3,5], the relationship between the two parameters was further examined in the present study. Linear regression analyses, based on the 18 pairs of either serum or tissue level of zinc and serum HDL cholesterol as obtained at 6 weeks, showed that the serum level of zinc was positively correlated (r = +0.65; P < 0.01) with serum HDL cholesterol (Fig. 1). No correlation (P > 0.05) was noted TABLE
5
EFFECTS OF EXPERIMENTS LIPOPROTEIN CHOLESTEROL
DIETS
ON THE
Serum lipids
Weeks after dietary 2nd (mg/lOO
SERUM
LEVELSa
OF TOTAL
LIPIDS
AND
treatment
ml)
6th (mg/lOO
Total triglyceride SAT UNS
61.0 f 4.3 a 55.7k4.2 a
84.9k33.7 b 84.9 f 8.9 b
Total cholesterol SAT UNS
99.3 f 9.7 a 97.1 f 9.4 B
110.1 f 9.7 a 91.3*7.4a
VLDL cholesterol SAT UNS
25.8 f 3.3 ’ 24.3 f 2.7 ’
32.2 f 3.0 ’ 27.3 f 4.2 a
LDL cholesterol SAT UNS
15.8 f 2.3 ’ 26.6 f 5.7 ’
18.8 f 1.6 a 17.8k2.1 ’
53.8 k5.5 a.b 46.2 f 3.7 b
59.1 f 6.4 ’ 46.2 f 3.2 b
ml)
HDL cholesterol SAT UNS
a Mean f SEM of 9 rats per group. Values in the same column common superscript are significantly different (P i 0.05).
or row for each parameter
not sharing
a
129
between the levels of tibia (or liver) zinc and HDL cholesterol. No relationships (P > 0.05) were indicated between serum (or tissue) concentrations of copper and any of the major lipoprotein fractions. Discussion
The hypocholesterolemic effect of dietary polyunsaturated fatty acid (PUFA) has been well-documented in animals as well as in humans (e.g. [12] and [13]). Such an effect of PUFA has been considered as beneficial with regard to reducing risk of CHD, although it still remains to be established whether the lowering of serum cholesterol levels by dietary means in general reduces the incidence of CHD [14]. In recent years, much attention has been directed toward the distribution of serum cholesterol among the major lipoprotein fractions as affected by dietary PUFA. Conflicting results have been reported as to whether the dietary PUFA-induced hypocholesterolemia is due to a decrease in VLDL- LDL-, and/or HDL cholesterol [12,13,15-191. The present data showed that the feeding of 0.6% linoleic acid for 6 weeks produced a significant decrease (22%) in serum HDL cholesterol, compared with a diet containing hydrogenated coconut oil with no supplemental linoleic acid. Under the present experimental conditions, dietary linoleic acid did not lower significantly the serum level of total cholesterol or the serum concentrations of VLDL- and LDL cholesterol. The levels of total serum cholesterol, however, tended to be lower in the group fed UNS diet at both 2 and 6 weeks, compared to the values for the rats fed SAT diet. It should also be pointed out that there appeared to be a transient rise in LDL cholesterol at 2 weeks ip the rats fed UNS diet (Table 5), although the increase was not statistically significant (P > 0.05). Such a response to dietary PUFA during an early phase of dietary treatment has been reported to occur, when the diet contains both cholesterol and unsaturated fat [30]. Previously, Durrington et al. [19] reported that, in healthy normolipidemic men, a high PUFA diet (P/S = 2.82) with a low cholesterol intake (112 mg/day) decreased the serum levels of total cholesterol by 18.7% compared with a low PUFA diet (P/S = 0.23) with a high cholesterol intake (667 mg/day), and that the decrease in total serum cholesterol was due primarily to a decrease in LDL cholesterol. In a similar study with normolipidemic subjects, Tan et al. [18] showed that, when compared to a high-saturated fat diet with a daily cholesterol intake of 1021 mg (P/S = 0.4), a high-PUFA, low-cholesterol (98 mg/day) diet decreased total serum cholesterol by lowering the cholesterol content in VLDL (59%) LDL (15%), and HDL (30%). In their recent series of human studies using a constant level of cholesterol (400 mg/day), Shepherd et al. showed that a high-PUFA diet (P/S = 4.0) lowered total plasma cholesterol by 23-24%, compared with a saturated fat diet (P/S = 0.25) by decreasing the cholesterol content in VLDL (25-27%), LDL (20-30%), and HDL (20-33%). In other recent animal [12] and human [16] studies, similar effects of dietary PUFA on all major lipoprotein fractions have been reported. In the present study, the dietary level of cholesterol was kept at 1% for both diets. The lowering of HDL cholesterol was observed before the hypocholesterolemic
130
effect of PUFA became clearly evident. Although the present results do not provide information concerning diet-induced changes in fatty acid composition of lipoproteins, previous studies [12,13,15,19] have demonstrated that the feeding of polyunsaturated fat increases the content of linoleic acid (C,sZ2) of lipoprotein lipids and decreases that of oleic acid (C,,: i) and palmitic acid (C,,:,). The degree of such changes is directly related to the extent of modification of the chemical and physical properties and hence metabolism (or clearance) rate of serum lipoproteins. The reason for the rather selective effect of PUFA on HDL cholesterol observed in the present experiment might be that the UNS diet contained a relatively small amount of linoleic acid with a low P/S ratio (0.18). Also, it might be explained by the general lack of response of the rat to diet-induced hypercholesterolemia. From the aforementioned and present studies, it is evident that the effects of dietary PUFA on serum lipoprotein levels depend upon the hypocholesterolemia induced and the concentration of PUFA relative to that of other dietary lipids, particularly, saturated fat and cholesterol. Based upon the above and present observations, the non-specific nature of PUFA effect in altering the levels of serum lipoproteins should be carefully interpreted. It is yet to be known whether the demonstrated effects of PUFA on the levels of total serum cholesterol and lipoproteins are beneficial in reducing risk of CHD. In the light of the suggested role of VLDL and LDL in atherogenesis [20,21] and well-documented inverse relationship between serum HDL level and incidence of CHD [22-281, it should be pointed out that the potential benefits of the hypocholesterolemic and LDL-lowering effects of PUFA must be evaluated against the suggested detrimental effect of a decrease in HDL produced by dietary PUFA [13,16,18]. In conjunction with the HDL-lowering effect of dietary PUFA, it is of particular interest to note that, in the present study, the serum level of zinc in the linoleic acid-fed rats were significantly lower at 6 weeks, relative to the level of zinc in the rats fed saturated fat. The data also show that the serum concentration of HDL cholesterol was significantly correlated (r = + 0.65; P -c0.01) with serum zinc status (Fig. 1). The phenomenon of positive correlation between serum zinc and HDL cholesterol has been first demonstrated in our previous studies using adult male rats depleted of zinc [5] and those fed cholesterol [3]. In the first study [5], we have shown that an acute depletion of zinc produces a selective decline in HDL cholesterol and that the serum status of zinc is strongly correlated (r = +0.81; P < 0.01) with serum HDL
Y -89.80 + 97.73.x r = + 0.65 PC 0.01
0: SAT 0: UNS
Serum zinc, ppm
1. Relationship
between
the serum levels of zinc and HDL cholesterol.
131
cholesterol. Subsequently [3], we have also observed that dietary cholesterol at 1% level significantly decreases the serum levels of both ‘zinc and HDL cholesterol and At that the two parameters are significantly correlated (r = + 0.57; P < 0.001). present, it is not known whether the zinc-lowering event mediated by dietary linoleic acid or dietary cholesterol is linked in a specific manner to the metabolism (e.g., synthesis and/or secretion) of HDL. In our previous studies [5,29] using zinc-depleted rats, we have postulated that the nutritional status of zinc may affect the synthesis of the apolipoproteins of chylomicrons and possibly of HDL. Based on the present findings, it is difficult to determine whether the rats fed linoleic acid were marginally deficient in zinc, although the levels of zinc in serum and tibia of the rats were significantly lowered by dietary linoleic acid. In the present study, the diets used were adequate in zinc (30 ppm), and the serum and tissue levels of zinc in these rats were within the ‘normal’ range. In addition, the food intake and body growth of the animals were not affected by the feeding of linoleic acid. However, the possibility that dietary linoleic acid may adversely affect the nutritional status of zinc has been suggested in a recent study using human subjects [4]. The study showed that, with the average zinc intake of 25 mg/day, the retentions of zinc effected by saturated and polyunsaturated fats were 5.9 and 0.6 mg/day, respectively, when the retention was determined by the intake minus fecal and urinary losses. This suggests that the ingestion of PUFA may influence the metabolism of zinc, perhaps interfering with the intestinal absorption and/or the turnover of zinc. The observations thus far made indicate that, for future studies on this subject, careful consideration should be given to the amounts and types of dietary lipids and their effects on the metabolism and body status of zinc. Further studies are underway to define the interactive effects of dietary triglyceride, cholesterol and zinc on the metabolism of HDL and zinc. In summary, the present study has shown that dietary PUFA (linoleic acid) at a relatively low level produces a decrease in serum HDL cholesterol without significantly lowering total serum cholesterol and that the decrease in HDL-cholesterol due to PUFA feeding is associated with the reduction in serum zinc. The practical significance of the zinc- and HDL-lowering effects of dietary PUFA remains to be evaluated. In addition, the phenomenon of the positive relationship between serum zinc and HDL cholesterol, as repeatedly observed in the present and previous studies [3,5], warrants further investigation into the possible biochemical role(s) of zinc in HDL metabolism. References 1 Mertz, W., Trace minerals and atherosclerosis, Fed. Proc., 41 (1982) 2807. 2 Underwood, E.J., I. Zinc in animal tissues and fluids. In: Trace Elements in Human and Animal Nutrition, 4th edition, Academic Press, New York, NY, 1977, pp. 196-205. 3 Koo, S.I. and Ramlet, J.S., Dietary cholesterol decreases the serum level of zinc - Further evidence for the positive relationship between serum zinc and high-density lipoproteins, Amer. J. Clin. Nutr., 37 (1983) 918. 4 Lukaski, H.C., Klevay, L.M., Bolonchuk, W.W. et al., Influence of dietary lipids on iron, zinc, and copper retention in trained athletes, Fed. Proc., 41 (1982) 275. 5 Koo, S.I. and Williams, D.A., Relationship between the nutritional status of zinc and cholesterol concentration of serum lipoproteins in adult male rats, Amer. J. Clin. Nutr., 34 (1981) 2376.
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Institute of Nutrition, Report of the American Institute of Nutrition Ad Hoc 6 The American Committee. on standards for nutritional studies, J. Nutr., 107 (1977) 1340. of orbital bleeding technic to rapid serial blood studies, Proc. Sot. Exp. Biol. 7 Riley, V., Adaptation Med., 104 (1960) 751. for measuring cholesterol in plasma lipopro8 Bronzert, T.J. and Brewer, Jr., H.B., New micromethod tein fractions, Clin. Chem., 23 (1977) 2089. M.F., Stone, P., Ellis, S. and Colwell, J.A., Cholesterol determination in high-density 9 Lopes-Virella, lipoproteins separated by three different methods, Clin. Chem., 23 (1977) 882. of total 10 Allain, C.C., Poon, L.S., Chan, C.S.G., Richmond, W. and Fu, P.C., Enzymatic determination serum cholesterol, Clin. Chem., 20 (1974) 470. determination of serum triglylcerides by the use of enzymes, 11 Bucolo, G. and David, H., Quantitative Clin. Chem., 19 (1973) 476. 12 Nicolosi, R.J., Marlett, J.A., Morello, A.M., Flanagan, S.A. and Hegsted, D.M., Influence of dietary unsaturated and saturated fat on the plasma lipoproteins of mongolian gerbils, Atherosclerosis, 38 (1980) 359. 13 Shepherd, J., Packard, C.J., Patsch, J.R., Gotto, Jr., A.M. and Taunton, O.D., Effects of dietary polyunsaturated and saturated fat on the properties of high density lipoproteins and the metabolism of apolipoprotein A-l, J. Clin. Invest., 61 (1978) 1582. 14 Food and Nutrition Board, National Research Council, Cardiovascular disease. In: Toward Healthful Diets, National Academy of Sciences, Washington, DC, 1980, pp. 8-12. 15 Shepherd, J., Packard, C.J., Grundy, SM. et al., Effects of saturated and polyunsaturated fat diets on the chemical composition and metabolism of low density lipoproteins in man, J. Lipid Res., 21 (1980) 91. 16 Vessby, B., Boberg, J., Gustafsson, I-B. et al., Reduction of high density lipoprotein cholesterol and apolipoprotein A-l concentrations by a lipid-lowering diet, Atherosclerosis, 35 (1980) 21. 17 Jackson, R.L., Taunton, O.D., Morrisett, J.D. and Gotto, Jr., A.M., The role of dietary polyunsaturated fat, Cir. Res., 42 (1978) 447. 18 Tan, M.H., Dickinson, M.A. and Albers, J.J., The effect of a high cholesterol and saturated fat diet on serum high-density lipoprotein-cholesterol, apoprotein A-l, and apoprotein E levels in normolipidemic humans, Amer. J. Clin. Nutr., 33 (1980) 2557. 19 Durrington, P.N., Bolton, C.H., Hartog, M., Angelinetta, R., Emmett, P. and Furniss, S., The effect of a low-cholesterol, high-polyunsaturate diet on serum lipid levels, apolipoprotein B levels and triglyceride fatty acid composition, Atherosclerosis, 27 (1977) 465. 20 Kannel, W.B., Castelli, W.P., Gordon, T. and McNamara, P.M., Serum cholesterol, lipoproteins, and the risk of coronary heart disease, Ann. Int. Med., 74 (1971) 1. 21 Camejo, G., Interaction of low density lipoproteins with arterial constituents - Its relationship with atherogenesis. In: C.E. Day and R.S. Levy (Eds.), Low Density Lipoproteins, Plenum Press, New York, NY, 1976, p. 351. 22 Carlson, L.A. and Ericsson, M., Quantitative and qualitative serum lipoprotein analysis, Part 2 (Studies in male survivors of myocardial infarction), Atherosclerosis, 21 (1975) 435. 23 Miller, N.E., Thelle, D.S., Ferde, O.H. and Mjos, O.D., The Tromso Heart Study, Lancet, i (1977) 965. 24 Rhoads, G.G., Gulbrandsen, C.L. and Kagan, A., Serum lipoprotein and coronary heart disease in a population study of Hawaii Japanese men, N. Engl. J. Med., 294 (1976) 293. 25 Castelli, W.P., Doyle, J.T., Gorden, T. et al., HDL-cholesterol and other lipids in coronary heart disease - The Cooperative Lipoprotein Phenotyping Study, Circulation, 55 (1977) 767. 26 Gordon, T., Castelli, W.P., Hjortland, M.J., Kannel, W.B. and Dawber, T.R., High density lipoprotein as a protective factor against coronary heart disease, Amer. J. Med., 62 (1977) 707. 27 Tyroler, H.A., Hames, C.G., Krishen, I., Hayden, S., Cooper, G. and Cassel, J.C., Black-white differences in serum lipids and lipoproteins in Evans County, Prevent. Med., 4 (1975) 541. 28 Miller, G.J. and Miller, N.E., Plasma-high-density-lipoprotein concentration and development of ischaemic heart-disease, Lancet, i (1975) 16. 29 Koo, S.I. and Turk, D.E., Effect of zinc deficiency on intestinal transport of triglycerides in the rat, J. Nutr., 107 (1977) 909. 30 Reiser, R., Clark, D.A., Sorrels, M.F., Gibson, B.S., Williams, M.C. and Wilson, F.H., Tissue cholesterol transport as modified by diet cholesterol and the nature of diet fat, J. Atheroscler. Res., 6 (1966) 565.