Journal of Nutrition and Metabolism

Journal of Nutrition and Metabolism / 2017 / Article

Research Article | Open Access

Volume 2017 |Article ID 4798963 | 10 pages | https://doi.org/10.1155/2017/4798963

A Moderate Zinc Deficiency Does Not Alter Lipid and Fatty Acid Composition in the Liver of Weanling Rats Fed Diets Rich in Cocoa Butter or Safflower Oil

Academic Editor: H. K. Biesalski
Received17 Oct 2016
Revised08 Mar 2017
Accepted19 Mar 2017
Published29 Mar 2017

Abstract

The aim of the study was to examine whether a moderate zinc deficiency alters hepatic lipid composition. Male weanling rats, assigned to five groups (8 animals each), were fed low-carbohydrate high-fat diets supplemented with 7 or 50 mg Zn/kg (LZ or HZ) and 22% cocoa butter (CB) or 22% safflower oil (SF) for four weeks. One group each had free access to the LZ-CB and LZ-SF diets, one group each was restrictedly fed the HZ-CB and HZ-SF diets in matching amounts, and one group had free access to the HZ-SF diet (ad libitum control). The rats fed the LZ diets had significantly lower energy intakes and final body weights than the ad libitum control group, and lower plasma and femur Zn concentrations than the animals consuming the HZ diets. Hepatic cholesterol, triacylglycerol and phospholipid concentrations, and fatty acid composition of hepatic triacylglycerols and phospholipids did not significantly differ between the LZ and their respective HZ groups, but were greatly affected by dietary fat source. In conclusion, the moderate Zn deficiency did not significantly alter liver lipid concentrations and fatty acid composition.

1. Introduction

Zinc (Zn) is the second most abundant trace element in the human and animal body. It is an essential cofactor for many hundreds of enzymes and numerous other proteins that fulfill a wide variety of biochemical processes in metabolism [1, 2]. Poor Zn status is considered to be one of the most common micronutrient deficiencies in human populations worldwide [3, 4]. Zn deficiency has been associated with many diseases, including diabetes, chronic liver disease, and cardiovascular disease [5]. Zn supplementation showed beneficial effects on plasma lipid parameters and may have the potential to reduce the incidence of atherosclerosis [5]. The impact of Zn deficiency on lipid metabolism has been studied extensively using rodents as models. Young animals can be readily depleted of zinc due to their high nutritional demands for growth, unlike adult humans [6]. Since Zn depletion of young animals causes anorexia and growth retardation, classical designs included limit-fed control animals in order to account for metabolic effects of reduced energy intake. Dietary energy restriction has been shown to effect significant changes in hepatic lipid composition under conditions of adequate Zn nutrition [7]. An alterative experimental paradigm widely used in Zn studies has been force-feeding young rats by gastric tube in order to equalize and synchronize food intake [811]. In these studies, Zn-depleted animals generally developed fatty livers and an altered fatty acid (FA) composition of liver lipids. In contrast, livers of rats given free access to Zn-deficient diets did not display increased triacylglycerol (TAG) concentrations compared with those of Zn-adequate controls [1215]. Most former studies are based on models of severe Zn deprivation. Marginal Zn deficiency is the more prevalent phenotype among human populations than clinical states of Zn depletion [5, 6].

The aim of our study was to investigate the effect of a moderate Zn depletion on hepatic lipid and FA composition in weanling rats fed diets rich in cocoa butter or safflower oil as sources of saturated and polyunsaturated FAs, respectively. Dietary fat source has been reported to interact with the effect of dietary Zn depletion on FA composition of hepatic lipids in young rats [9, 10, 15]. High-fat diets are apt to foster FA oxidation for maintenance and growth [16]. A mild Zn deficit allows a significant accretion of lean tissue including membrane lipids during the growth spurt in the postweaning period, whereas severe Zn depletion leads to growth arrest by impairing cell division and proliferation [2].

2. Methods

2.1. Animals and Experimental Design

A total of 40 male weanling Wistar rats (Harlan-Winkelmann, Borchen, Germany) with an initial body weight of  g (mean ± SD) were divided into five groups of eight animals each. These groups were randomly assigned to one of four semisynthetic diets that were supplemented with 7.0 or 50 mg zinc as Zn sulfate per kg (LZ and HZ diets, resp.) and with either cocoa butter (CB) or safflower oil (SF). Dietary treatments of the groups were as follows: LZ-CB, fed the LZ-CB diet free choice, HZ-CBR, fed the HZ-CB diet in restricted amounts equal to intake in the LZ-CB group on the previous day, LZ-SF, fed the LZ-SF diet free choice, HZ-SFR, fed the HZ-SF diet in restricted amounts equal to intake in the LZ-SF group on the previous day, and HZ-SF, fed the HZ-SF diet free choice (ad libitum control). An ad libitum-fed HZ-CB control group was not included because dietary fat source did not affect food intake and growth of weanling rats fed Zn-adequate diets free choice in our former experiment [15]. All animals had free access to demineralized water. They were kept individually in metabolic cages (stainless-steel metal grids) under controlled environmental conditions (22°C, 60% rel. humidity, 12 h dark-light cycle). Food remainders were removed daily and weighed. During wk 3 and 4, faeces were collected quantitatively from each animal and stored at −20°C until analysis. After four weeks, food was withdrawn overnight for 10 to 12 h before the animals were anesthetized in a carbon dioxide atmosphere and killed by decapitation. Blood was collected in heparinized tubes to prepare plasma by centrifugation (10 min at 1500 ×g). The liver and right femur bone were removed from the carcasses and stored at −80°C. All experimental treatments of the rats followed established guidelines for the care and handling of laboratory animals. Approval was obtained by the Animal Protection Authority of the State (II 25.3-19c20/15c GI 19/3).

2.2. Diets

All diets contained, per kg, 200 g powdered egg albumen, 67 g corn starch, 100 g sucrose, 280 g cellulose, 30 g soybean oil, 3.0 g lysine plus methionine (1 : 1 by wt.), 100 g mineral plus vitamin premixes [17], and 220 g cocoa butter or 220 g safflower oil. They were stored at 4°C after preparation. The high cellulose addition served to restrict the dietary energy density. Except for the fat components, the dietary metabolizable (ME) contents are based on tabulated values of the ingredients [18]. Fat digestibility of the CB and SF diets, assessed during wk 3 and 4, significantly differed () (Figure 1) and averaged (overall mean ± SD) () and % (), respectively. On the basis of 39 kJ/g digestible fat, the CB and SF diets were estimated to contain 15.1 and 16.9 kJ ME per gram dry matter, respectively. Carbohydrates (starch plus sucrose) and fat contributed 24.3 and 55.0% of the ME in the CB diets, and 21.7 and 59.9% in the SF diets. The LZ-CB, LZ-SF, HZ-CB, and HZ-SF diets contained, by analysis (mean ± SD, ), , , , and μg Zn/g dry matter, respectively. Based on energy density, Zn concentrations in the LZ-CB and LZ-SF diets were 0.517 and 0.462 μg/kJ ME, respectively. Table 1 presents the analytical FA composition of the diets.


Fatty acidsCB dietsSF diets

Palmitic (16:0)26.37.9
Palmitoleic (16:1)0.20.1
Stearic (18:0)29.02.3
Oleic (18:1n-9)31.612.5
Linoleic (18:2n-6)10.575.3
α-Linolenic (18:3n-3)1.21.1
SFAs56.410.7
MUFAs31.912.9
PUFAs11.776.4
PUFA/SFA ratio0.217.14
n-6/n-3 ratio8.868.5
Relative UI0.571.67

Contain very long-chain SFAs.
Contain very long-chain MUFAs.
SFAs, saturated fatty acids; MUFAs, monounsaturated fatty acids; PUFAs, polyunsaturated fatty acids; UI, unsaturation index.
2.3. Analytical Methods
2.3.1. Zn Analyses

Plasma samples were diluted with 0.1 M HCl and analyzed for zinc by atomic absorption spectrometry (PU 9400, Phillips, Kassel, Germany). Samples of diets, livers, and femur bones were wet-ashed with 65% (w/vol) HNO3 for 16 h, and appropriately diluted with aqua bidest for Zn analysis by ICP-AES (Type 701, Unicam). Zn analyses were replicated at least twice per sample, and accuracy was validated by the analysis of standard samples of known Zn concentration.

2.3.2. Lipid Analyses

The fat content of diets and faeces (collected in wk 3 and 4) was analyzed by an official method [19] for the determination of fat digestibility. Faeces were treated with 4 M HCl before fat extraction.

Liver concentrations of total lipids, cholesterol, TAGs, and phospholipids (PLs) were determined in duplicate lipid extracts as previously described [15]. Precision and accuracy of cholesterol and TAG assays were assessed with Qualitrol (Merck, Darmstadt, Germany).

2.3.3. FA Analyses

Diet samples were heated in 0.4 M HCl and dried before lipid extraction by n-hexane (0.005% BHT). Liver PLs and TAGs were isolated from lipid extracts [15] by solid-phase extraction [20]. Briefly, hepatic lipid extracts were vacuum-dried, redissolved in chloroform-isopropanol (C-I, 2 : 1, by vol), and fractionated with hexane-conditioned aminopropyl Bond Elut columns (Bond Elut NH2 500, Agilent Technologies). Neutral lipids were eluted with C-I, free FAs with 2% acetic acid (by vol) in diethylether, and the polar PLs with methanol. The neutral lipid fraction was redissolved in n-hexane after removal of the C-I eluant and transferred to fresh Bond Elut columns to elute cholesteryl esters with n-hexane and finally to elute TAGs with n-hexane containing 1% diethylether and 10% dichloromethane (by vol). PL and TAG fractions were vacuum-dried, redissolved in isopropanol (0.005% BHT), and stored at −80°C for FA analysis.

Lipid extracts of diets and liver PLs and TAGs fractions were supplemented with 1,2,3-triheptadecanoylglycerol as internal standard, condensed at 45°C under a nitrogen stream, redissolved in n-hexane (0.005% BHT), and transmethylated with N-trimethylsulfoniumhydroxide (Macherey & Nagel) at room temperature [21]. The FA methylesters (FAMEs) were separated and quantified by a GLC system (Chrompack 9400) that was equipped with an autosampler, a 50 m Permabond FFAP-DF column (Macherey & Nagel), and a flame ionization detector. Hydrogen was used as carrier gas. FAMEs were separated in a temperature gradient program and identified on the basis of their retention times compared to an authentic FAME mix (C4–C24, number 18919-1AMP Supelco, Sigma-Aldrich) that was supplemented with three additional polyenoic FAs (cis-13,16,19-docosatrienoic acid, Sigma; cis-7,10,13,16-docosatetraenoic acid, Sigma; cis-7,10,13,16,19-docosapentaenoic acid, Supelco). FAME peak areas were quantified in relation to the peak areas of the internal standard (heptadecanoic acid). The relative unsaturation index (UI) of FAs in diets, liver TAGs, and PLs was calculated by multiplying the molar percentages of FAs by the number of double bonds present and dividing the total sum of products by hundred [22].

2.4. Statistical Analyses

The data of the five diet groups were statistically analyzed by using SPSS for Windows (version 19; IBM). Homogeneity of variance was checked by Levene’s test. In the case of heterogeneity of variance, data were logarithmically transformed. Data of the five groups were subjected to one-factor ANOVA, followed by Tukey’s multiple-comparison test (the level of significance being set at ). In addition, results of the LZ-CB, HZ-CBR, LZ-SF, and HZ-SFR groups were analyzed by bifactorial ANOVA to test for main effects of Zn, fat (fat source), and Zn × fat interaction. Correlations are based on Pearson’s correlation coefficients.

3. Results

3.1. Food and Energy Intake, Final Body Weights, and Zn Status of the Rats

Food and ME intake and final body weights of the weanling rats fed the LZ-CB and LZ-SF diets free choice were comparable to those of the rats fed the corresponding HZ diets in equivalent amounts but markedly lower than those recorded for the animals fed the HZ-SF diet free choice (Table 2). Dietary fat source also significantly affected food and ME intake and final body weights, which were at least 20% lower in the LZ-SF than in the LZ-CB group. ME intake per gram of body weight gain was similar among groups except for a significantly () higher value in the case of the LZ-SF group. Zn intake per gram body weight gain did not differ between the LZ-CB and LZ-SF groups (), but was higher () in the HZ-CBR group than in the HZ-SFR and -SF groups.


Diet groups2 × 2 ANOVA: P level
LZ-CBHZ-CBRLZ-SFHZ-SFRHZ-SFZnFatZn × fat

Food intakeg DM/d0.70<0.0010.44
ME intakekJ/d0.73<0.0010.45
ME intakekJ/g BWG0.005<0.0010.052
Zn intakeμg/g BWG<0.0010.0010.062
Final body weightsg0.31<0.0010.21

LZ-CB, low-Zn (7.8 μg Zn/g DM) cocoa butter (CB) diet fed free choice; HZ-CBR, high-Zn (53 μg Zn/g DM) CB diet fed in restricted amounts equivalent to the intake of the LZ-CB diet; LZ-SF, low-Zn (7.8 μg Zn/g DM) safflower oil (SF) diet fed free choice; HZ-SFR, high-Zn (53 μg Zn/g DM) SF diet fed in restricted amounts equivalent to the intake of the LZ-SF diet; HZ-SF, HZ-SF diet fed free choice.
Values are means ± SD, . Labeled means in a row without a common letter differ by 1-factor ANOVA followed by Tukey’s multiple-comparison test, .
Bifactorial ANOVA of the LZ-CB, HZ-CBR, LZ-SF, and HZ-SFR diet groups.
1-factor and bifactorial ANOVA after logarithmic transformation of the data.
BWG, body weight gain; DM, dry matter; ME, metabolizable energy.

Plasma and femur Zn concentrations were greatly reduced in the LZ-CB and LZ-SF groups compared with the HZ groups (Figure 2). Furthermore, the rats fed the LZ- and HZ-SF diets had lower () plasma and femur Zn concentrations than the animals fed the corresponding LZ- and HZ-CB diets. In contrast, liver Zn concentrations were not affected by dietary treatments ().

3.2. Liver Lipid Concentrations

The dietary Zn level did not significantly alter hepatic concentrations of total lipids, cholesterol, TAGs, and PLs (Table 3). But livers of the rats fed the SF diets had significantly () higher concentrations of cholesterol and TAGs than those consuming the CB diets. The highest total lipid and TAG concentrations were recorded in the rats fed the HZ-SF diet free choice. CB-fed rats had approximately 10% higher PL concentrations than the SF-fed animals ().


Diet groups2 × 2 ANOVA: P level
LZ-CBHZ-CBRLZ-SFHZ-SFRHZ-SFZnFatZn × fat

Total lipidsmg/g0.160.0030.77
Cholesterolμmol/g0.08<0.0010.86
TAGsμmol/g0.25<0.0010.37
PLsμmol/g0.990.0010.81

See footnote 1 of Table 2.
Values are means ± SD, . Labeled means in a row without a common letter differ by 1-factor ANOVA followed by Tukey’s multiple-comparison test, .
Bifactorial ANOVA of the LZ-CB, HZ-CBR, LZ-SF, and HZ-SFR diet groups.
1-factor ANOVA after logarithmic transformation of the data.
Bifactorial ANOVA after logarithmic transformation of the data.
TAGs, triacylglycerols; PLs, phospholipids.
3.3. FA Composition of Liver PLs

The bifactorial ANOVA of the FA composition of hepatic PLs displays significant () Zn effects in the case of palmitic acid (16:0), dihomo-γ-linolenic acid (20:3n-6), arachidonic acid (20:4n-6), total n-6 polyunsaturated FAs (n-6 PUFAs), and the ratios n-6/n-3 PUFAs (Table 4). These effects mainly result from differences between the LZ-SF and HZ-SFR groups. Dietary fat source markedly affected the proportions of all FAs except for palmitic acid. Saturated FAs (SFAs) accounted for 43 and 40 mol%, and n-6 PUFAs for about 40 and 50% of the total FAs in the CB and SF groups, respectively. In all five diet groups, arachidonic acid was the most abundant PUFA of PLs and contributed at least one-third of the total FAs. Molar proportions of n-3 PUFAs were approximately threefold higher than the proportions of monounsaturated FAs (MUFAs). Docosahexaenoic acid (22:6n-3) was the prevailing n-3 PUFA and present in significantly () greater amounts in the CB than in the SF groups. Total Δ6 desaturation products of linoleic (18:2n-6) and α-linolenic acid (18:3n-3) account for approximately 90% of the total PUFAs in the CB-fed rats and for 80% in the SF-fed animals. The ratios of n-6/n-3 PUFAs averaged 3.2 in the two CB groups, whereas they were approximately twice as high in the three SF groups. The relative UI of the liver PLs, however, was closely comparable among the five diet groups ().


Fatty acidsDiet groups2 × 2 ANOVA: P level
LZ-CBHZ-CBRLZ-SFHZ-SFRHZ-SFZnFatZn × fat

16:00.0030.590.56
18:00.85<0.0010.14
SFAs0.14<0.0010.12
18:1n-90.99<0.0010.056
MUFAs 0.59<0.0010.10
18:2n-60.53<0.0010.84
20:3n-60.0010.520.050
20:4n-60.012<0.0010.40
22:4n-60.150.62<0.0010.65
n-6 PUFAs60.022<0.0010.19
22:5n-30.420.0280.73
22:6n-350.21<0.0010.34
n-3 PUFAs50.17<0.0010.30
Δ6 DS products70.75<0.0010.52
Ratio n-6/n-30.017<0.0010.029
Relative UI0.410.0670.64

See footnote 1 of Table 2.
Values are means ± SD, . Labeled means in a row without a common letter differ by 1-factor ANOVA followed by Tukey’s multiple-comparison test, .
Bifactorial ANOVA of the LZ-CB, HZ-CBR, LZ-SF, and HZ-SFR diet groups.
Contain 16:1, 20:1n-9, and 22:1n-9.
1-factor and bifactorial ANOVA after logarithmic transformation of the data.
Contain 20:2n-6.
Total Δ6 desaturation products (mol per 100 mol PUFAs) contain 20:3n-6, 20:4n-6, 22:4n-6, 22:5n-3, and 22:n-3.
SFAs, saturated fatty acids; MUFAs, monounsaturated fatty acids; PUFAs, polyunsaturated fatty acids; UI, relative unsaturation index.
3.4. FA Composition of Liver TAGs

The FA composition of liver TAGs shows significant () Zn effects on the molar proportions of SFAs, largely because of differences between the LZ-CB and HZ-CBR groups (Table 5), which accordingly affect the relative UI. Proportions of MUFAs and PUFAs were not significantly () altered by the dietary Zn level. TAGs of the HZ-SFR and HZ-SF groups contained closely comparable proportions of total SFAs, MUFAs, and PUFAs. Dietary fat source, however, greatly modified the FA pattern. The abundance of SFAs in the CB groups was nearly three times as high as in the SF groups (). Oleic acid (18:1n-9) was the prevailing single FA in TAGs of the CB-fed rats, whereas linoleic acid dominated in TAGs of the SF-fed animals. The proportions of n-3 PUFAs did not exceed 2.5 and 1.5 mol% in the CB- and SF-fed animals, respectively. The ratios of n-6/n-3 PUFAs in the SF groups were approximately eightfold and the relative UI twofold higher than in the CB groups.


Fatty acidsDiet groups2 × 2 ANOVA: P level
LZ-CBHZ-CBRLZ-SFHZ-SFRHZ-SFZnFatZn × fat

16:0<0.001<0.0010.018
18:00.021<0.0010.48
SFAs 0.006<0.0010.17
18:1n-90.098<0.0010.044
MUFAs 0.16<0.0010.058
18:2n-60.41<0.0010.52
18:3n-60.72<0.0010.088
20:4n-640.051<0.0010.70
22:4n-60.088<0.0010.056
n-6 PUFAs70.072<0.0010.65
18:3n-30.700.0360.81
22:5n-30.90<0.0010.11
22:6n-30.055<0.0010.15
n-3 PUFAs 0.31<0.0010.77
Δ6 DS products80.280.0010.96
Ratio n-6/n-35